Methods and systems for characterizing analytes from individual cells or cell populations

ABSTRACT

The present disclosure provides methods of processing or analyzing a sample. A method for processing a sample may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule), barcoding the probe-nucleic acid molecule complex, and performing extension, denaturation, and amplification processes. A method for processing a sample may comprise hybridizing first and second probes to adjacent or non-adjacent target regions of a nucleic acid molecule (e.g., an RNA molecule), linking the first and second probes to provide a probe-linked nucleic acid molecule, and barcoding the probe-linked nucleic acid molecule. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well. One or more processes of the methods described herein may be performed on a cell, such as a permeabilized cell.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.17/680,209, filed Feb. 24, 2022, which is a continuation of U.S.application Ser. No. 17/229,557, filed, Apr. 13, 2021, which is acontinuation application of U.S. application Ser. No. 16/554,564, filedAug. 28, 2019, which is a continuation-in-part of U.S. Application No.PCT/US2019/019309, filed Feb. 22, 2019, which claims the benefit of U.S.Provisional Patent Application No. 62/804,648, filed Feb. 12, 2019, andU.S. Provisional Patent Application No. 62/633,982, filed Feb. 22, 2018,each of which applications are entirely incorporated herein byreference.

BACKGROUND

Samples may be processed for various purposes, such as identification ofa type of moiety within the sample. The sample may be a biologicalsample. The biological samples may be processed for various purposes,such as detection of a disease (e.g., cancer) or identification of aparticular species. There are various approaches for processing samples,such as polymerase chain reaction (PCR) and sequencing.

Biological samples may be processed within various reactionenvironments, such as partitions. Partitions may be wells or droplets.Droplets or wells may be employed to process biological samples in amanner that enables the biological samples to be partitioned andprocessed separately. For example, such droplets may be fluidicallyisolated from other droplets, enabling accurate control of respectiveenvironments in the droplets.

Partitions and/or biological samples in partitions may be subjected tovarious processes, such as chemical processes or physical processes.Partitions and/or samples in partitions may be subjected to heating orcooling, or chemical reactions, such as to yield species that may bequalitatively or quantitatively processed.

SUMMARY

The present disclosure provides methods for use in various sampleprocessing and analysis applications. The methods provided herein mayinvolve hybridizing a probe to a target region of a nucleic acidmolecule of interest, barcoding the resultant complex, and performing anextension, denaturation, and amplification processes to provide nucleicacid molecules comprising a sequence the same or substantially the sameas or complementary to that of the target region of the nucleic acidmolecule of interest. A method may comprise hybridizing a first probeand a second probe to first and second target regions of the nucleicacid molecule, linking the first and second probes to provide aprobe-linked nucleic acid molecule, and barcoding the probe-linkednucleic acid molecule. One or more processes of the methods providedherein may be performed within a partition such as a droplet or well.The methods of the present disclosure may obviate the need for reversetranscription during analysis of ribonucleic acid molecules and may beuseful, for example, in controlled analysis and processing of analytessuch as biological particles, nucleic acids, and proteins.

In an aspect, provided herein is a method of analyzing a samplecomprising a nucleic acid molecule, comprising: (a) providing: (i) asample comprising the nucleic acid molecule, wherein the nucleic acidmolecule comprises a first target region and a second target region,wherein the first target region is adjacent to the second target region;(ii) a first probe comprising a first probe sequence and a second probesequence, wherein the first probe sequence of the first probe iscomplementary to the first target region of the nucleic acid molecule,and wherein the first probe sequence comprises a first reactive moiety;and (iii) a second probe comprising a third probe sequence, wherein thethird probe sequence of the second probe is complementary to the secondtarget region of the nucleic acid molecule, and wherein the third probesequence comprises a second reactive moiety; (b) subjecting the sampleto conditions sufficient to (i) hybridize the first probe sequence ofthe first probe to the first target region of the nucleic acid molecule,and (ii) hybridize the third probe sequence of the second probe to thesecond target region of the nucleic acid molecule, such that the firstreactive moiety of the first probe sequence of the first probe isadjacent to the second reactive moiety of the third probe sequence ofthe second probe; (c) subjecting the first reactive moiety and thesecond reactive moiety to conditions sufficient to yield a probe-linkednucleic acid molecule comprising the first probe linked to the secondprobe; and (d) with the probe-linked nucleic acid molecule in apartition, barcoding the probe-linked nucleic acid molecule to provide abarcoded probe-linked nucleic acid molecule.

In some embodiments, the partition is a well. In some embodiments thepartition is a droplet. In some embodiments, (d) comprises (i)providing, in the partition, a nucleic acid barcode molecule comprisinga binding sequence and a barcode sequence, wherein the binding sequenceis complementary to the second probe sequence of the first probe, and(ii) hybridizing the binding sequence to the second probe sequence inthe partition. In some embodiments, the nucleic acid barcode moleculefurther comprises an additional binding sequence. In some cases, thebinding sequence is hybridized to the second probe sequence in apartition among a plurality of partitions. In some embodiments,subsequent to (c), the probe-linked nucleic acid molecule isco-partitioned with the nucleic acid barcode molecule. In someembodiments, subsequent to (a), the nucleic acid molecule isco-partitioned with the first probe, the second probe, and the nucleicacid barcode molecule. In some embodiments, (b) and (c) are performed inthe partition. In some embodiments, the method further comprisessubjecting the partition to conditions sufficient to conduct anamplification reaction using the barcoded probe-linked nucleic acidmolecule, thereby generating an amplification product within thepartition. In some embodiments, the amplification reaction is apolymerase chain reaction. In some embodiments, the method furthercomprises releasing the amplification product from the partition. Insome embodiments, the method further comprises sequencing theamplification product.

In some embodiments, the second probe comprises a fourth probe sequence,and wherein (d) further comprises providing, in the partition, a nucleicacid binding molecule, wherein the nucleic acid binding moleculecomprises a second binding sequence that is complementary to the fourthprobe sequence of the second probe. In some embodiments, the nucleicacid binding molecule further comprises a third binding sequence. Insome embodiments, the nucleic acid binding molecule further comprises asecond barcode sequence. In some embodiments, the method furthercomprises hybridizing the second binding sequence to the fourth probesequence of the second probe in the partition.

In some embodiments, the nucleic acid barcode molecule is attached to abead. In some embodiments, the bead is a gel bead. In some embodiments,the bead comprises a plurality of nucleic acid barcode moleculesattached thereto, wherein the plurality of nucleic acid barcodemolecules comprise the nucleic acid barcode molecule. In someembodiments, the bead comprises at least 10,000 nucleic acid barcodemolecules attached thereto. In some embodiments, the bead comprises atleast 100,000 nucleic acid barcode molecules attached thereto. In someembodiments, the bead comprises at least 1,000,000 nucleic acid barcodemolecules attached thereto. In some embodiments, the bead comprises atleast 10,000,000 nucleic acid barcode molecules attached thereto. Insome embodiments, the plurality of nucleic acid barcode molecules arereleasably attached to the bead. In some embodiments, the plurality ofnucleic acid barcode molecules are releasable from the bead uponapplication of a stimulus. In some embodiments, the stimulus is selectedfrom the group consisting of a thermal stimulus, a photo stimulus, and achemical stimulus. In some embodiments, the stimulus is a reducingagent. In some embodiments, the stimulus is dithiothreitol.

In some embodiments, the application of the stimulus results in one ormore of (i) cleavage of a linkage between nucleic acid barcode moleculesof the plurality of nucleic acid barcode molecules and the bead, and(ii) degradation of the bead to release nucleic acid barcode moleculesof the plurality of nucleic acid barcode molecules from the bead. Insome embodiments, (d) comprises (i) providing, in the partition, thenucleic acid barcode molecule releasably attached to the bead, whereinthe nucleic acid barcode molecule comprises the binding sequence and thebarcode sequence; (ii) releasing the nucleic acid barcode molecule fromthe bead; and (iii) hybridizing the binding sequence of the releasednucleic acid barcode molecule to the second probe sequence in thepartition.

In some embodiments, the first probe further comprises a barcodesequence or unique molecular identifier. In some embodiments, the secondprobe further comprises a barcode sequence or a unique molecularidentifier.

In some embodiments, the first reactive moiety of the first probecomprises an azide moiety. In some embodiments, the second reactivemoiety of the second probe comprises an alkyne moiety. In someembodiments, the first probe is linked to the second probe in theprobe-linked nucleic acid molecule via a linker, wherein the linkercomprises a triazole moiety.

In some embodiments, the first reactive moiety of the first probecomprises a phosphorothioate moiety. In some embodiments, the secondreactive moiety of the second probe comprises an iodide moiety. In someembodiments, the first probe is linked to the second probe in theprobe-linked nucleic acid molecule via a linker, wherein the linkercomprises a phosphorothioate bond.

In some embodiments, the first reactive moiety of the first probecomprises an amine moiety. In some embodiments, the second reactivemoiety of the second probe comprises a phosphate moiety. In someembodiments, the first probe is linked to the second probe in theprobe-linked nucleic acid molecule via a linker, wherein the linkercomprises a phosphoramidate bond.

In some embodiments, the first reactive moiety of the first probecomprises an amine moiety. In some embodiments, the second reactivemoiety of the second probe comprises a phosphate moiety. In someembodiments, the first probe is linked to the second probe in theprobe-linked nucleic acid molecule via a linker, wherein the linkercomprises a phosphoroamidate bond. In some embodiments, the samplecomprises a cell, and wherein the nucleic acid molecule is containedwithin the cell. In some embodiments, the method further comprises,subsequent to (a), permeabilizing the cell, thereby providing access tothe nucleic acid molecule. The cell may be alive or dead (e.g., fixed).In some embodiments, the method further comprises, subsequent to (a),lysing the cell, thereby releasing the nucleic acid molecule from thecell. In some embodiments the cell is a prokaryotic cell. In someembodiments, the cell is a eukaryotic cell. In some embodiments, thecell is a lymphocyte. In some embodiments, the cell is a B cell. In someembodiments, the cell is a T cell. In some embodiments, the cell is ahuman cell. In some embodiments, the cell is provided within thepartition.

In some embodiments, the nucleic acid molecule is a single-strandednucleic acid molecule. In some embodiments, the nucleic acid moleculecomprises a polyA sequence at a terminus of the nucleic acid molecule.In some embodiments, the nucleic acid molecule comprises an untranslatedregion (UTR). In some embodiments, the nucleic acid molecule comprises a5′ cap structure. In some embodiments, the nucleic acid molecule is aribonucleic acid (RNA) molecule. In some embodiments, the nucleic acidmolecule is a messenger RNA (mRNA) molecule.

In some embodiments, the nucleic acid molecule is a deoxyribonucleicacid (DNA) molecule.

In some embodiments, the partition further comprises one or morereagents selected from the group consisting of fluorophores,oligonucleotides, primers, nucleic acid barcode molecules, barcodes,buffers, deoxynucleotide triphosphates, DNA splints, detergents,reducing agents, chelating agents, oxidizing agents, nanoparticles,antibodies, and enzymes.

In some embodiments, the partition further comprises one or morereagents selected from the group consisting of temperature-sensitiveenzymes, pH-sensitive enzymes, light-sensitive enzymes, proteases,ligase, polymerases, restriction enzymes, nucleases, proteaseinhibitors, and nuclease inhibitors.

In some embodiments, the sample comprises a cell bead, and wherein thenucleic acid molecule is contained within the cell bead.

In some embodiments, (a)-(c) are performed without reversetranscription.

In some embodiments, the first probe and the second probe are parts ofthe same nucleic acid molecule.

In another aspect, provided herein is a method of analyzing a samplecomprising a nucleic acid molecule, comprising: (a) providing: (i) asample comprising the nucleic acid molecule, wherein the nucleic acidmolecule comprises a first target region, a gap region, and a secondtarget region, wherein the gap region is disposed between the firsttarget region and the second target region; (ii) a first probecomprising a first probe sequence and a second probe sequence, whereinthe first probe sequence of the first probe is complementary to thefirst target region of the nucleic acid molecule; and (iii) a secondprobe comprising a third probe sequence, wherein the third probesequence of the second probe is complementary to the second targetregion of the nucleic acid molecule; (b) subjecting the sample toconditions sufficient to (i) hybridize the first probe sequence of thefirst probe to the first target region of the nucleic acid molecule,(ii) hybridize the third probe sequence of the second probe to thesecond target region of the nucleic acid molecule, and (iii) yield aprobe-linked nucleic acid molecule comprising the first probe linked tothe second probe; and (c) with the probe-linked nucleic acid molecule ina partition, barcoding the probe-linked nucleic acid molecule to providea barcoded probe-linked nucleic acid molecule.

In some embodiments, the partition is a well. In some embodiments, thepartition is a droplet.

In some embodiments, (b) comprises performing a nucleic acid reaction.In some embodiments, (b) comprises performing an enzymatic ligationreaction or an extension reaction. In some embodiments, (b) comprisesperforming the extension reaction and the enzymatic ligation reaction.In some embodiments, the nucleic acid reaction comprises using an enzymeselected from the group consisting of T4 RNL2, KOD ligase, SplintR,PBCV1, DNA polymerase, and Mu polymerase, or a derivative thereof. Insome embodiments, the gap region comprises a length of at least onebase.

In some embodiments, (c) comprises (i) providing, in the partition, anucleic acid barcode molecule comprising a binding sequence and abarcode sequence, wherein the binding sequence is complementary to thesecond probe sequence of the first probe, and (ii) hybridizing thebinding sequence to the second probe sequence in the partition. In someembodiments, the nucleic acid barcode molecule further comprises anadditional binding sequence. In some embodiments, the binding sequenceis hybridized to the second probe sequence in a partition among aplurality of partitions.

In some embodiments, the method further comprises, subsequent to (b),co-partitioning the probe-linked nucleic acid molecule and the nucleicacid barcode molecule. In some embodiments, subsequent to (a), thenucleic acid molecule is co-partitioned with the first probe, the secondprobe, and the nucleic acid barcode molecule. In some embodiments, (b)is performed in the partition. In some embodiments, the method furthercomprises subjecting the partition to conditions sufficient to conductan amplification reaction using the barcoded probe-linked nucleic acidmolecule, thereby generating an amplification product within thepartition. In some embodiments, the amplification reaction is apolymerase chain reaction. In some embodiments, the method furthercomprises releasing the amplification product from the partition. Insome embodiments, the method further comprises sequencing theamplification product. In some embodiments, the second probe comprises afourth probe sequence, and wherein (d) further comprises providing, inthe partition, a nucleic acid binding molecule, wherein the nucleic acidbinding molecule comprises a second binding sequence that iscomplementary to the fourth probe sequence of the second probe.

In some embodiments, the nucleic acid binding molecule further comprisesa third binding sequence. In some embodiments, the nucleic acid bindingmolecule further comprises a second barcode sequence. In someembodiments, the method further comprises hybridizing the second bindingsequence to the fourth probe sequence of the second probe in thepartition. In some embodiments, the nucleic acid barcode molecule isattached to a bead. In some embodiments, the bead is a gel bead. In someembodiments, the bead comprises a plurality of nucleic acid barcodemolecules attached thereto, wherein the plurality of nucleic acidbarcode molecules comprise the nucleic acid barcode molecule. In someembodiments, the bead comprises at least 10,000 nucleic acid barcodemolecules attached thereto. In some embodiments, the bead comprises atleast 100,000 nucleic acid barcode molecules attached thereto. In someembodiments, the bead comprises at least 1,000,000 nucleic acid barcodemolecules attached thereto. In some embodiments, the bead comprises atleast 10,000,000 nucleic acid barcode molecules attached thereto. Insome embodiments, the plurality of nucleic acid barcode molecules arereleasably attached to the bead. In some embodiments, the plurality ofnucleic acid barcode molecules are releasable from the bead uponapplication of a stimulus. In some embodiments, the stimulus is selectedfrom the group consisting of a thermal stimulus, a photo stimulus, and achemical stimulus. In some embodiments, the stimulus is a reducingagent. In some embodiments, the stimulus is dithiothreitol.

In some embodiments, the application of the stimulus results in one ormore of (i) cleavage of a linkage between nucleic acid barcode moleculesof the plurality of nucleic acid barcode molecules and the bead, and(ii) degradation of the bead to release nucleic acid barcode moleculesof the plurality of nucleic acid barcode molecules from the bead.

In some embodiments, (c) comprises (i) providing, in the partition, thenucleic acid barcode molecule releasably attached to the bead, whereinthe nucleic acid barcode molecule comprises the binding sequence and thebarcode sequence; (ii) releasing the nucleic acid barcode molecule fromthe bead; and (iii) hybridizing the binding sequence of the releasednucleic acid barcode molecule to the second probe sequence in thepartition. In some embodiments, the first probe further comprises abarcode sequence or unique molecular identifier. In some embodiments,the second probe further comprises a barcode sequence or a uniquemolecular identifier. In some embodiments, the sample comprises a cell,and wherein the nucleic acid molecule is contained within the cell. Insome embodiments, the method further comprises, subsequent to (a),permeabilizing the cell, thereby providing access to the nucleic acidmolecule. In some embodiments, the method further comprises, subsequentto (a), lysing the cell, thereby releasing the nucleic acid moleculefrom the cell.

In some embodiments, the cell is a prokaryotic cell. In someembodiments, the cell is a eukaryotic cell. In some embodiments, thecell is a lymphocyte. In some embodiments, the cell is a B cell. In someembodiments, the cell is a T cell. In some embodiments, the cell is ahuman cell. In some embodiments, the cell is provided within thepartition. In some embodiments, the nucleic acid molecule is asingle-stranded nucleic acid molecule. In some embodiments, the nucleicacid molecule comprises a polyA sequence at a terminus of the nucleicacid molecule.

In some embodiments, the nucleic acid molecule comprises an untranslatedregion (UTR). In some embodiments, the nucleic acid molecule comprises a5′ cap structure. In some embodiments, the nucleic acid molecule is aribonucleic acid (RNA) molecule. In some embodiments, the nucleic acidmolecule is a messenger RNA (mRNA) molecule. In some embodiments, thenucleic acid molecule is a deoxyribonucleic acid (DNA) molecule.

In some embodiments, the partition further comprises one or morereagents selected from the group consisting of fluorophores,oligonucleotides, primers, nucleic acid barcode molecules, barcodes,buffers, deoxynucleotide triphosphates, ribonucleoside triphosphates,DNA splints, detergents, reducing agents, chelating agents, oxidizingagents, nanoparticles, antibodies, and enzymes.

In some embodiments, the partition further comprises one or morereagents selected from the group consisting of temperature-sensitiveenzymes, pH-sensitive enzymes, light-sensitive enzymes, proteases,ligase, polymerases, restriction enzymes, nucleases, proteaseinhibitors, and nuclease inhibitors.

In some embodiments, the sample comprises a cell bead, and the nucleicacid molecule is contained within the cell bead. In some embodiments,(b) is performed without reverse transcription.

In some embodiments, the first probe or the second probe comprises aknown sequence.

In some embodiments, the first probe or the second probe comprises adegenerate sequence.

In some embodiments, the first probe or the second probe comprises aPhi-29 based rolling circle amplification sequence.

In another aspect, provided herein is a method of analyzing a samplecomprising a nucleic acid molecule, comprising: (a) providing: (i) asample comprising the nucleic acid molecule, wherein the nucleic acidmolecule comprises a target region; (ii) a probe comprising a probesequence and an adapter sequence, wherein the probe sequence iscomplementary to the target region; and (iii) an adapter comprising abinding sequence, wherein the binding sequence is complementary to theadapter sequence; (b) subjecting the sample to conditions sufficient to(i) hybridize the probe sequence of the probe to the target region, and(ii) hybridize the adapter sequence of the probe to the binding sequenceof the adapter, to yield an adapter-bound probe; and (c) with theadapter-bound probe in a partition, barcoding the adapter-bound probe toprovide a barcoded nucleic acid molecule.

In some embodiments, the adapter sequence comprises between 5 to 10nucleotides.

In another aspect, the present disclosure provides a method of analyzinga sample comprising a nucleic acid molecule, comprising: (a) providing:(i) a sample comprising the nucleic acid molecule, wherein the nucleicacid molecule comprises a first target region, a gap region, and asecond target region, wherein the gap region is disposed between thefirst target region and the second target region; (ii) a first probecomprising a first probe sequence and a second probe sequence, whereinthe first probe sequence of the first probe is complementary to thefirst target region of the nucleic acid molecule; and (iii) a secondprobe comprising a third probe sequence, wherein the third probesequence of the second probe is complementary to the second targetregion of the nucleic acid molecule; (b) subjecting the sample toconditions sufficient to (i) hybridize the first probe sequence of thefirst probe to the first target region of the nucleic acid molecule,(ii) hybridize the third probe sequence of the second probe to thesecond target region of the nucleic acid molecule, and (iii) yield aprobe-linked nucleic acid molecule comprising the first probe linked tothe second probe; and (d) with the probe-linked nucleic acid molecule ina partition, barcoding the probe-linked nucleic acid molecule to providea barcoded probe-linked nucleic acid molecule.

In another aspect, the present disclosure provides a method of analyzinga sample comprising a nucleic acid molecule, comprising: (a) providing:(i) a sample comprising said nucleic acid molecule, wherein said nucleicacid molecule comprises a first target region and a second targetregion, wherein said first target region and said second target regionare disposed on a same strand of said nucleic acid molecule; (ii) afirst probe comprising a first probe sequence and a second probesequence, wherein said first probe sequence of said first probe iscomplementary to said first target region of said nucleic acid molecule;and (iii) a second probe comprising a third probe sequence, wherein saidthird probe sequence of said second probe is complementary to saidsecond target region of said nucleic acid molecule; (b) subjecting saidsample to conditions sufficient to (i) hybridize said first probesequence of said first probe to said first target region of said nucleicacid molecule, and (ii) hybridize said third probe sequence of saidsecond probe to said second target region of said nucleic acid moleculeto yield a probe-associated nucleic acid molecule; (c) subjecting saidprobe-associated nucleic acid molecule to conditions sufficient to yielda probe-linked nucleic acid molecule comprising said first probe linkedto said second probe; and (d) within a partition, attaching a barcodesequence to said probe-linked nucleic acid molecule.

In some embodiments, said partition is a well among a plurality ofwells.

In some embodiments, said partition is a droplet among a plurality ofdroplets.

In some embodiments, (d) comprises (i) providing, in said partition, anucleic acid barcode molecule comprising a binding sequence and abarcode sequence, wherein said binding sequence is complementary to saidsecond probe sequence of said first probe, and (ii) subjecting saidpartition to conditions sufficient to hybridize said binding sequence tosaid second probe sequence. In some embodiments, the method furthercomprises subjecting said partition to conditions sufficient to conducta nucleic acid extension reaction to generate a barcoded nucleic acidmolecule comprising a sequence corresponding to said first probe, asequence corresponding to said second probe, and a sequencecorresponding to said barcode sequence. In some embodiments, the methodfurther comprises subjecting said partition to conditions sufficient toligate said probe-linked nucleic acid molecule to said nucleic acidbarcode molecule to generate a barcoded nucleic acid molecule comprisinga sequence corresponding to said first probe, a sequence correspondingto said second probe, and a sequence corresponding to said barcodesequence. In some embodiments, the method further comprises subjectingsaid barcoded nucleic acid molecule to conditions sufficient to conductan amplification reaction to generate an amplification product, whichamplification product comprises nucleic acid molecules comprising saidsequence corresponding to said first probe, said sequence correspondingto said second probe, and said sequence corresponding to said barcodesequence. In some embodiments, the amplification reaction comprises useof a primer comprising one or more functional sequences and wherein saidamplification product comprises nucleic acid molecules furthercomprising said one or more functional sequences. In some embodiments,said amplification is isothermal amplification. In some embodiments,said amplification reaction is performed within said partition. In someembodiments, the method further comprises recovering said amplificationproduct from said partition. In some embodiments, said amplificationreaction is performed outside of said partition. In some embodiments,the method further comprises sequencing said amplification product or aderivative thereof.

In some embodiments, the method further comprises (i) providing a splintoligonucleotide comprising a first sequence complementary to said secondprobe sequence and a second sequence, and (ii) subjecting said partitionto conditions sufficient to hybridize said first sequence of said splintoligonucleotide to said second probe sequence. In some embodiments, saidfirst sequence of said splint oligonucleotide hybridizes to said secondprobe sequence prior to (c). In some embodiments, said first sequence ofsaid splint oligonucleotide hybridizes to said second probe sequenceafter (c). In some embodiments, (d) comprises (i) providing, in saidpartition, a nucleic acid barcode molecule comprising a binding sequenceand a barcode sequence, wherein said binding sequence is complementaryto said second sequence of said splint oligonucleotide, and (ii)subjecting said partition to conditions sufficient to hybridize saidbinding sequence to said second sequence of said splint oligonucleotide.In some embodiments, said binding sequence of said nucleic acid barcodemolecule comprises one or more ribobases. In some embodiments, saidmethod further comprises subjecting (i) said splint oligonucleotidehybridized to said second probe sequence and (ii) said nucleic acidbarcode molecule to conditions sufficient to ligate said probe-linkednucleic acid molecule to said nucleic acid barcode molecule. In someembodiments, said method further comprises subjecting (i) said splintoligonucleotide hybridized to said second probe sequence and (ii) saidnucleic acid barcode molecule to conditions sufficient to conduct anucleic acid extension reaction to generate a barcoded nucleic acidmolecule comprising a sequence corresponding to said first probe, asequence corresponding to said second probe, and a sequencecorresponding to said barcode sequence. In some embodiments, said methodfurther comprises subjecting said barcoded nucleic acid molecule toconditions sufficient to conduct an amplification reaction to generatean amplification product, which amplification product comprises nucleicacid molecules comprising said sequence corresponding to said firstprobe, said sequence corresponding to said second probe, and saidsequence corresponding to said barcode sequence. In some embodiments,said amplification reaction is a polymerase chain reaction. In someembodiments, said amplification reaction is performed within saidpartition. In some embodiments, said method further comprises recoveringsaid amplification product from said partition. In some embodiments,said amplification reaction is performed outside of said partition. Insome embodiments, said amplification reaction is isothermalamplification. In some embodiments, the method further comprisessequencing said amplification product or derivative thereof.

In some embodiments, said nucleic acid barcode molecule furthercomprises a unique molecular identifier sequence, a sequencing primersequence, and/or a partial sequencing primer sequence. In someembodiments, subsequent to (c), said probe-associated nucleic acidmolecule is co-partitioned with said nucleic acid barcode molecule. Insome embodiments, subsequent to (a), said nucleic acid molecule isco-partitioned with said first probe, said second probe, and saidnucleic acid barcode molecule. In some embodiments, (c) is performedwithin said partition. In some embodiments, (b) and (c) are performedwithin said partition.

In some embodiments, said second probe comprises a fourth probesequence, and wherein said method further comprises providing a nucleicacid binding molecule in said partition, wherein said nucleic acidbinding molecule comprises a second binding sequence that iscomplementary to said fourth probe sequence of said second probe. Insome embodiments, the method further comprises hybridizing said secondbinding sequence to said fourth probe sequence of said second probewithin said partition.

In some embodiments, said nucleic acid barcode molecule is coupled to abead. In some embodiments, said bead is a gel bead. In some embodiments,said nucleic acid barcode molecule is coupled to said bead via a labilemoiety. In some embodiments, said bead comprises a plurality of nucleicacid barcode molecules coupled thereto, wherein said plurality ofnucleic acid barcode molecules comprise said nucleic acid barcodemolecule. In some embodiments, said bead comprises at least 100,000nucleic acid barcode molecules coupled thereto. In some embodiments,said plurality of nucleic acid barcode molecules are releasably coupledto said bead. In some embodiments, said plurality of nucleic acidbarcode molecules are releasable from said bead upon application of astimulus. In some embodiments, said stimulus is selected from the groupconsisting of a thermal stimulus, a photo stimulus, a biologicalstimulus, and a chemical stimulus. In some embodiments, said stimulus isa reducing agent. In some embodiments, the application of said stimulusresults in one or more of (i) cleavage of a linkage between nucleic acidbarcode molecules of said plurality of nucleic acid barcode moleculesand said bead, and (ii) degradation of said bead to release nucleic acidbarcode molecules of said plurality of nucleic acid barcode moleculesfrom said bead. In some embodiments, said bead is provided in saidpartition, and wherein said nucleic acid barcode molecule is releasedfrom said bead within said partition.

In some embodiments, (c) is performed before (d). In some embodiments,(d) is performed before (c).

In some embodiments, said first probe or said second probe furthercomprises a barcode sequence or unique molecular identifier.

In some embodiments, said second probe comprises a fourth probesequence, which fourth probe sequence hybridizes to a third targetregion of said nucleic acid molecule. In some embodiments, said secondtarget region is not adjacent to said third target region, and whereinsaid third probe sequence and said fourth probe sequence of said secondprobe are separated by a linker sequence.

In some embodiments, said first probe sequence of said first probecomprises a first reactive moiety and said third probe sequence of saidsecond probe comprises a second reactive moiety, wherein, subsequent to(b), said first reactive moiety is adjacent to said second reactivemoiety. In some embodiments, wherein (c) comprises subjecting said firstreactive moiety and said second reactive moiety to conditions sufficientto link said first probe sequence to said third probe sequence. In someembodiments, said first reactive moiety of said first probe or saidsecond reactive moiety of said second probe comprises an azide moiety,an alkyne moiety, a phosphorothioate moiety, an iodide moiety, an aminemoiety, or a phosphate moiety. In some embodiments, said first probe islinked to said second probe in said probe-linked nucleic acid moleculevia a linker, wherein said linker comprises a triazole moiety, aphosphorothioate bond, or a phosphoroamidatephosphoramidate bond.

In some embodiments, (c) comprises performing an enzymatic ligationreaction and/or an extension reaction. In some embodiments, saidenzymatic ligation reaction and/or said extension reaction comprises useof an enzyme selected from the group consisting of T4 RNL2, SplintR, T4DNA ligase, KOD ligase, PBCV1, DNA polymerase, and Mu polymerase, or aderivative thereof. In some embodiments, prior to (a), said first probeis linked to said second probe via one or more linking sequences. Insome embodiments, said one or more linking sequences comprise one ormore of a spacer sequence, a sequencing primer or complement thereof, acapture sequence, a restriction site, a transposition site, and a uniquemolecular identifier sequence. In some embodiments, said one or morelinking sequences comprise a thermolabile, photocleavable, orenzymatically cleavable site.

In some embodiments, said first target region is adjacent to said secondtarget region.

In some embodiments, said first target region and said second targetregion are separated by a gap region disposed between said first targetregion and said second target region. In some embodiments, said gapregion is at least one nucleotide long. In some embodiments, said gapregion is at least 10 nucleotides long. In some embodiments, said gapregion is at least 100 nucleotides long.

In some embodiments, the method further comprises digesting one or morenucleic acid molecules or portions thereof using an exonuclease.

In some embodiments, said first probe or said second probe comprises aknown sequence or a degenerate sequence.

In some embodiments, said first probe or said second probe comprises aPhi-29 based rolling circle amplification sequence. In some embodiments,said first probe or said second probe comprises a cleavable site,wherein said cleavable site is cleavable using a thermal, photo-,chemical, or biological stimulus. In some embodiments, the methodfurther comprises contacting said first probe or said second probe witha transposase.

In some embodiments, said sample comprises a cell, and wherein saidnucleic acid molecule is contained within said cell. In someembodiments, the method further comprises, subsequent to (a), lysing orpermeabilizing said cell, thereby providing access to said nucleic acidmolecule. In some embodiments, said cell is a prokaryotic cell. In someembodiments, said cell is a eukaryotic cell. In some embodiments, saidcell is a human cell. In some embodiments, said cell is a fixedsuspension cell or a formalin-fixed paraffin-embedded cell. In someembodiments, said cell is provided within said partition. In someembodiments, said cell is a single cell.

In some embodiments, said nucleic acid molecule is a ribonucleic acid(RNA) molecule. In some embodiments, said nucleic acid molecule is amessenger RNA (mRNA) molecule. In some embodiments, said nucleic acidmolecule comprises a poly-A sequence at a terminus of said nucleic acidmolecule.

In some embodiments, said nucleic acid molecule is a deoxyribonucleicacid (DNA) molecule.

In some embodiments, said partition further comprises one or morereagents selected from the group consisting of fluorophores,oligonucleotides, primers, nucleic acid barcode molecules, barcodes,buffers, deoxynucleotide triphosphates, DNA splints, detergents,reducing agents, chelating agents, oxidizing agents, nanoparticles,antibodies, temperature-sensitive enzymes, pH-sensitive enzymes,light-sensitive enzymes, proteases, ligases, polymerases, reversetranscriptases, restriction enzymes, nucleases, protease inhibitors, andnuclease inhibitors. In some embodiments, said polymerase is apolymerase selected from the group of DNA polymerase, RNA polymerase,Hot Start polymerase, and Warm start polymerase. In some embodiments,said sample comprises a cell bead, and wherein said nucleic acidmolecule is contained within said cell bead. In some embodiments,(a)-(c) are performed without reverse transcription.

In yet another aspect, the present disclosure provides a method ofanalyzing a sample comprising a nucleic acid molecule, comprising: (a)providing: (i) a sample comprising said nucleic acid molecule, whereinsaid nucleic acid molecule comprises a target region; (ii) a probecomprising a probe sequence and a binding sequence, wherein said probesequence is complementary to said target region; and (iii) an adaptercomprising a first sequence and a second sequence, wherein said firstsequence of said adapter is complementary to said binding sequence ofsaid probe; (b) subjecting said sample to conditions sufficient tohybridize (i) said probe sequence of said probe to said target region,and (ii) said binding sequence of said probe to said first sequence ofsaid adapter, to yield an adapter-bound probe; and (c) within apartition, barcoding said adapter-bound probe to provide a barcodednucleic acid molecule.

In a further aspect, the present disclosure provides a method ofanalyzing a sample comprising a nucleic acid molecule, comprising: (a)providing: (i) a sample comprising said nucleic acid molecule, whereinsaid nucleic acid molecule comprises a target region; (ii) a probecomprising a probe sequence and a first reactive moiety, wherein saidprobe sequence is complementary to said target region; and (iii) anucleic acid barcode molecule comprising a second reactive moiety and abarcode sequence; (b) subjecting said sample to conditions sufficient tohybridize said probe sequence of said probe to said target region toprovide a probe-associated nucleic acid molecule; and (c) within apartition, subjecting said first reactive moiety of saidprobe-associated nucleic acid molecule and said second reactive moietyof said nucleic acid barcode molecule to conditions sufficient to linksaid probe-associated nucleic acid molecule and said nucleic acidbarcode molecule to provide a barcoded nucleic acid product.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual biological particles.

FIG. 2 shows an example of a microfluidic channel structure fordelivering barcode carrying beads to droplets.

FIG. 3 shows an example of a microfluidic channel structure forco-partitioning biological particles and reagents.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.FIG. 7B shows a perspective view of the channel structure of FIG. 7A.

FIG. 8 illustrates an example of a barcode carrying bead.

FIG. 9 schematically illustrates a method for analyzing a target nucleicacid molecule. Panel 9A illustrates a probe hybridized to a targetnucleic acid molecule. Panel 9B illustrates a nucleic acid barcodemolecule hybridized to a sequence of the probe and Panel 9C illustratesextension of the probe. Panel 9D illustrates optional denaturation of anextended nucleic acid molecule from the target nucleic acid molecule.Panel 9E illustrates amplification of the extended nucleic acidmolecule.

FIG. 10 schematically illustrates a method for analyzing a targetnucleic acid molecule. Panel 10A illustrates a target nucleic acidmolecule, a first probe, and a second probe, and Panel 10B illustrates atarget nucleic acid molecule with the first and second probes hybridizedthereto. Panel 10C illustrates a probe-linked nucleic acid molecule,while Panel 10D illustrates a barcoded probe-linked nucleic acidmolecule.

FIG. 11 illustrates a barcoding scheme using a split-pool approach.Panel 11A illustrates a probe-bound nucleic acid molecule. Panel 11Bshows the addition of a first barcode sequence segment. Panel 11C showsthe addition of a second barcode sequence segment. Panel 11D showsaddition of a third barcode sequence segment.

FIG. 12 schematically illustrates a method of analyzing a target nucleicacid molecule. Panel 12A illustrates a target nucleic acid molecule, afirst probe, and a second probe, and Panel 12B illustrates a targetnucleic acid molecule with the first and second probes hybridizedthereto. Panel 12C illustrates a probe-linked nucleic acid molecule,while Panel 12D illustrates a barcode molecule hybridized to aprobe-linked nucleic acid molecule. Panel 12E illustrates a barcodedprobe-linked nucleic acid molecule.

FIGS. 13A-13B schematically illustrate a method of analyzing a targetnucleic acid molecule using a molecular inversion probe. FIG. 13Aschematically illustrates a method of using such a probe. Panel 13Aillustrates a molecular inversion probe comprising first and secondprobe ends hybridized to a target nucleic acid molecule. Panel 13Billustrates a circular probe-linked nucleic acid molecule. Panel 13Cillustrates cleavage and linearization of the circular probe forbarcoding. FIG. 13B illustrates circularization of a first probe and asecond probe using a splint molecule.

FIG. 14 shows a sample workflow for analysis of a plurality of nucleicacid molecules involving co-partitioning nucleic acid molecules withbarcoded beads within droplets.

FIG. 15 shows various approaches for chemically-mediated nucleic acidligation.

Panel 15A illustrates formation of a triazole bond. Panel 15Billustrates formation of a phosphorothioate bond. Panel 15C illustratesformation of an amide bond. Panel 15D illustrates a formation ofphosphoramidate bond. Panel 15E illustrates a conjugation reaction.

FIG. 16 shows a method for analyzing a nucleic acid molecule. Panel 16Aillustrates a target nucleic acid molecule, a first probe, and a secondprobe, while Panel 16B illustrates a nucleic acid molecule with thefirst and second probes hybridized thereto and extension of the gapbetween probes. Panel 16C illustrates an extended nucleic acid molecule,and Panel 16D illustrates a probe-linked nucleic acid molecule.

FIG. 17 illustrates a method for analyzing a target nucleic acidmolecule. Panel 17A shows a target nucleic acid molecule and a firstprobe. Panel 17B illustrates a target nucleic acid molecule with thefirst probe hybridized thereto and a hybridization of an adaptor nucleicacid molecule to a sequence of the probe. Panel 17C illustrateshybridization of a barcode nucleic acid molecules to the adaptor nucleicacid molecule to generate a barcoded nucleic acid molecule.

FIG. 18 schematically shows a method of analyzing a nucleic acidmolecule.

FIG. 19 schematically shows another example method of analyzing anucleic acid molecule.

FIG. 20 schematically illustrates a method of analyzing a nucleic acidmolecule.

FIG. 20A schematically shows barcoding of a nucleic acid molecule. Panel20A illustrates a probe-linked nucleic acid molecule, while Panel 20Billustrates a splint molecule associated with a probe-linked nucleicacid molecule. Panel 20C illustrates a nucleic acid barcode moleculeassociating with the adapter molecule associated with a probe-linkednucleic acid molecule while Panel 20D illustrates a barcodedprobe-linked nucleic acid molecule.

FIG. 21 schematically illustrates a method of analyzing a nucleic acidmolecule. Panel 21A illustrates a nucleic acid molecule, a first probe,a second probe, and Panel 21B illustrates a nucleic acid molecule withthe first and second probes hybridized thereto. Panel 21C illustrates abarcoded nucleic acid molecule, while Panel 21D illustrates digestion ofunhybridized nucleic acid molecules. Panel 21E illustrates aprobe-linked, barcoded nucleic acid molecule.

FIG. 22A-C illustrates a method for multiplexed barcoding.

FIG. 23 shows a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

FIG. 24 illustrates an exemplary method for multiplexed barcoding.

FIG. 25 illustrates an exemplary method for multiplexed barcoding.

FIG. 26 illustrates an exemplary method for multiplexed barcoding.

FIG. 27 illustrates an exemplary method for multiplexed barcoding.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte. A barcode can be part of an analyte. A barcode can beindependent of an analyte. A barcode can be a tag attached to an analyte(e.g., nucleic acid molecule) or a combination of the tag in addition toan endogenous characteristic of the analyte (e.g., size of the analyteor end sequence(s)). A barcode may be unique. Barcodes can have avariety of different formats. For example, barcodes can include:polynucleotide barcodes; random nucleic acid and/or amino acidsequences; and synthetic nucleic acid and/or amino acid sequences. Abarcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads.

The terms “barcode nucleic acid molecule” and “nucleic acid barcodemolecule” may be used interchangeably herein. A barcode nucleic acidmolecule may comprise a barcode. A barcode nucleic acid molecule mayalso comprise adapters, such as a unique molecular identifier sequence.

The term “real time,” as used herein, can refer to a response time ofless than about 1 second, a tenth of a second, a hundredth of a second,a millisecond, or less. The response time may be greater than 1 second.In some instances, real time can refer to simultaneous or substantiallysimultaneous processing, detection or identification.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. For example, the subject can be a vertebrate, a mammal, arodent (e.g., a mouse), a primate, a simian or a human. Animals mayinclude, but are not limited to, farm animals, sport animals, and pets.A subject can be a healthy or asymptomatic individual, an individualthat has or is suspected of having a disease (e.g., cancer) or apre-disposition to the disease, and/or an individual that is in need oftherapy or suspected of needing therapy. A subject can be a patient. Asubject can be a microorganism or microbe (e.g., bacteria, fungi,archaea, viruses).

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject's hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions (e.g., that code for proteins) as well as non-coding regions. Agenome can include the sequence of all chromosomes together in anorganism. For example, the human genome ordinarily has a total of 46chromosomes. The sequence of all of these together may constitute ahuman genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. The terms “adapter”, “adapter molecule”, and “adapternucleic acid sequence” may also be used interchangeably herein. Anadaptor or tag can be coupled to a polynucleotide sequence to be“tagged” by any approach, including ligation, hybridization, or otherapproaches. An adapter molecule, in some cases, may be any usefulnucleic acid sequence and may include, for example, a sequencing primersite, a barcode sequence, a transposition site, a restriction site, aunique molecular identifier, a binding sequence, and any/or derivatives,variations, or combinations thereof.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example, nucleicacid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), including variants or derivatives thereof (e.g., single strandedDNA). Sequencing can be performed by various systems currentlyavailable, such as, without limitation, a sequencing system byIllumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or LifeTechnologies (Ion Torrent®). Alternatively or in addition, sequencingmay be performed using nucleic acid amplification, polymerase chainreaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR),or isothermal amplification. Such systems may provide a plurality of rawgenetic data corresponding to the genetic information of a subject(e.g., human), as generated by the systems from a sample provided by thesubject. In some examples, such systems provide sequencing reads (also“reads” herein). A read may include a string of nucleic acid basescorresponding to a sequence of a nucleic acid molecule that has beensequenced. In some situations, systems and methods provided herein maybe used with proteomic information.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead.The gel bead may include a polymer matrix (e.g., matrix formed bypolymerization or cross-linking). The polymer matrix may include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Polymers in the polymer matrix may be randomly arranged,such as in random copolymers, and/or have ordered structures, such as inblock copolymers. Cross-linking can be via covalent, ionic, orinductive, interactions, or physical entanglement. The bead may be amacromolecule. The bead may be formed of nucleic acid molecules boundtogether. The bead may be formed via covalent or non-covalent assemblyof molecules (e.g., macromolecules), such as monomers or polymers. Suchpolymers or monomers may be natural or synthetic. Such polymers ormonomers may be or include, for example, nucleic acid molecules (e.g.,DNA or RNA). The bead may be formed of a polymeric material. The beadmay be magnetic or non-magnetic. The bead may be rigid. The bead may beflexible and/or compressible. The bead may be disruptable ordissolvable. The bead may be a solid particle (e.g., a metal-basedparticle including but not limited to iron oxide, gold or silver)covered with a coating comprising one or more polymers. Such coating maybe disruptable or dissolvable.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may comprise any number ofmacromolecules, for example, cellular macromolecules. The sample may bea cell sample. The sample may be a cell line or cell culture sample. Thesample can include one or more cells. The sample can include one or moremicrobes. The biological sample may be a nucleic acid sample or proteinsample. The biological sample may also be a carbohydrate sample or alipid sample. The biological sample may be derived from another sample.The sample may be a tissue sample, such as a biopsy, core biopsy, needleaspirate, or fine needle aspirate. The sample may be a fluid sample,such as a blood sample, urine sample, or saliva sample. The sample maybe a skin sample. The sample may be a cheek swab. The sample may be aplasma or serum sample. The sample may be a cell-free or cell freesample. A cell-free sample may include extracellular polynucleotides.Extracellular polynucleotides may be isolated from a bodily sample thatmay be selected from the group consisting of blood, plasma, serum,urine, saliva, mucosal excretions, sputum, stool and tears.

The term “biological particle,” as used herein, generally refers to adiscrete biological system derived from a biological sample. Thebiological particle may be a macromolecule. The biological particle maybe a small molecule. The biological particle may be a virus. Thebiological particle may be a cell or derivative of a cell. Thebiological particle may be an organelle. The biological particle may bea rare cell from a population of cells. The biological particle may beany type of cell, including without limitation prokaryotic cells,eukaryotic cells, bacterial, fungal, plant, mammalian, or other animalcell type, mycoplasmas, normal tissue cells, tumor cells, or any othercell type, whether derived from single cell or multicellular organisms.The biological particle may be a constituent of a cell. The biologicalparticle may be or may include DNA, RNA, organelles, proteins, or anycombination thereof. The biological particle may be or may include amatrix (e.g., a gel or polymer matrix) comprising a cell or one or moreconstituents from a cell (e.g., cell bead), such as DNA, RNA,organelles, proteins, or any combination thereof, from the cell. Thebiological particle may be obtained from a tissue of a subject. Thebiological particle may be a hardened cell. Such hardened cell may ormay not include a cell wall or cell membrane. The biological particlemay include one or more constituents of a cell, but may not includeother constituents of the cell. An example of such constituents is anucleus or an organelle. A cell may be a live cell. The live cell may becapable of being cultured, for example, being cultured when enclosed ina gel or polymer matrix, or cultured when comprising a gel or polymermatrix.

A cell bead may include a single cell or a plurality of cells, or aderivative of the single cell or multiple cells. For example afterlysing and washing the cells, inhibitory components from cell lysatescan be washed away and the macromolecular constituents can be bound ascell beads. Systems and methods disclosed herein can be applicable toboth cell beads (and/or droplets or other partitions) containingbiological particles and cell beads (and/or droplets or otherpartitions) containing macromolecular constituents of biologicalparticles. In some cases, a cell or a plurality of cells may be alive,and the cells may be subjected to further processing, e.g., celllabeling.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within or from a biological particle. Themacromolecular constituent may comprise a nucleic acid. In some cases,the biological particle may be a macromolecule. The macromolecularconstituent may comprise DNA. The macromolecular constituent maycomprise RNA. The RNA may be coding or non-coding. The RNA may bemessenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), forexample. The RNA may be a transcript. The RNA may be small RNA that areless than 200 nucleic acid bases in length, or large RNA that aregreater than 200 nucleic acid bases in length. Small RNAs may include5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA(miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs),Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and smallrDNA-derived RNA (srRNA). The RNA may be double-stranded RNA orsingle-stranded RNA. The RNA may be circular RNA. The macromolecularconstituent may comprise a protein. The macromolecular constituent maycomprise a peptide. The macromolecular constituent may comprise apolypeptide.

The term “molecular tag,” as used herein, generally refers to a moleculecapable of binding to a macromolecular constituent. The molecular tagmay bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity. The molecular tag may comprise a nucleotide sequence. Themolecular tag may comprise a nucleic acid sequence. The nucleic acidsequence may be at least a portion or an entirety of the molecular tag.The molecular tag may be a nucleic acid molecule or may be part of anucleic acid molecule. The molecular tag may be an oligonucleotide or apolypeptide. The molecular tag may comprise a DNA aptamer. The moleculartag may be or comprise a primer. The molecular tag may be, or comprise,a protein. The molecular tag may comprise a polypeptide. The moleculartag may be a barcode.

The term “partition,” as used herein, generally, refers to a space orvolume that may be suitable to contain one or more species or conductone or more reactions. A partition may be a physical compartment, suchas a droplet or well (e.g., a microwell). The partition may isolatespace or volume from another space or volume. The droplet may be a firstphase (e.g., aqueous phase) in a second phase (e.g., oil) immisciblewith the first phase. The droplet may be a first phase in a second phasethat does not phase separate from the first phase, such as, for example,a capsule or liposome in an aqueous phase. A partition may comprise oneor more other (inner) partitions. In some cases, a partition may be avirtual compartment that can be defined and identified by an index(e.g., indexed libraries) across multiple and/or remote physicalcompartments. For example, a physical compartment may comprise aplurality of virtual compartments.

Provided herein are methods that may be used for various sampleprocessing and/or analysis applications. A method of the presentdisclosure may allow barcoding a nucleic acid molecule (e.g., aribonucleic acid (RNA) molecule) within a partition without performingreverse transcription. The nucleic acid molecule barcoded may be atargeted nucleic acid molecule. Such a method may involve attaching aprobe to the nucleic acid molecule, and subsequently attaching a nucleicacid barcode molecule comprising a barcode sequence to the probe. Forexample, the nucleic acid barcode molecule may attach to an overhangingsequence of the probe or to the end of the probe. Extension from an endof the probe to an end of the nucleic acid barcode molecule may form anextended nucleic acid molecule comprising both a sequence complementaryto the barcode sequence and a sequence complementary to a target regionof the nucleic acid molecule. The extended nucleic acid molecule maythen be denatured from the nucleic acid barcode molecule and the nucleicacid molecule and duplicated. This method may avoid the use of reversetranscription, which may be highly error prone. One or more processes ofthe method may be carried out within a partition such as a droplet orwell.

The present disclosure also provides a method of processing a samplethat provides a barcoded nucleic acid molecule having linked probemolecules attached thereto. The method may comprise one or moreligation-mediated reactions. The method may comprise providing a samplecomprising a nucleic acid molecule (e.g., an RNA molecule) havingadjacent first and second target regions; a first probe having a firstprobe sequence that is complementary to the first target region and asecond probe sequence; and a second probe having a third probe sequencethat is complementary to the second target region. The first and thirdprobe sequences may also comprise first and second reactive moieties,respectively. Upon hybridization of the first probe sequence of thefirst probe to the first target region of the nucleic acid molecule, andhybridization of the third probe sequence of the second probe to thesecond target region of the nucleic acid molecule, the reactive moietiesmay be adjacent to one another. Subsequent reaction between the adjacentreactive moieties under sufficient conditions may link the first andsecond probes to yield a probe-linked nucleic acid molecule. Theprobe-linked nucleic acid molecule may also be referred to as aprobe-ligated nucleic acid molecule. The probe-linked nucleic acidmolecule may then be barcoded with a barcode sequence of a nucleic acidbarcode molecule to provide a barcoded probe-linked nucleic acidmolecule. Barcoding may be achieved by hybridizing a binding sequence ofthe nucleic acid barcode molecule to the second probe sequence of thefirst probe of the probe-linked nucleic acid molecule. The barcodedprobe linked-nucleic acid molecule may be subjected to amplificationreactions to yield an amplified product comprising the first and secondtarget regions and the barcode sequence or sequences complementary tothese sequences. Accordingly, the method may provide amplified productswithout the use of reverse transcription. One or more processes may beperformed within a partition such as a droplet or well.

Further provided herein are methods of processing a sample that providesa barcoded nucleic acid molecule having linked probe molecules attachedthereto. The method may comprise one or more nucleic acid reactions. Themethod may comprise providing a sample comprising a nucleic acidmolecule (e.g., an RNA molecule) having first and second target regionson a same strand (e.g., adjacent or non-adjacent target regions); afirst probe having a first probe sequence that is complementary to thefirst target region and a second probe sequence; and a second probehaving a third probe sequence that is complementary to the second targetregion. The third probe sequence may be known or degenerate (i.e.,randomly generated). The first and third probe sequences may alsocomprise first and second reactive moieties, respectively. Where thenucleic acid molecule has non-adjacent first and second target regions,the nucleic acid molecule may comprise one or more gap regions betweenthe first and second target regions. Upon hybridization of the firstprobe sequence of the first probe to the first target region of thenucleic acid molecule, and the third probe sequence of the second probeto the second target region of the nucleic acid molecule, to yield aprobe-associated nucleic acid molecule, the reactive moieties may beadjacent or non-adjacent to one another. Subsequent reaction between theadjacent or non-adjacent probes may generate a probe-linked nucleic acidmolecule. The probe-linked nucleic acid molecule may also be referred toherein as a probe-ligated nucleic acid molecule. The probe-linkednucleic acid molecule may then be barcoded with a barcode sequence of anucleic acid barcode molecule to provide a barcoded probe-linked nucleicacid molecule. Barcoding may be achieved by hybridizing a bindingsequence of the nucleic acid barcode molecule to the second probesequence of the first probe of the probe-linked nucleic acid molecule.Barcoding may also be achieved by hybridizing a binding sequence of abarcode nucleic acid molecule to a nucleic acid adaptor sequence, wherethe nucleic acid adaptor sequence comprises a binding sequence that canhybridize to one or more nucleic acid probes. The barcoded probelinked-nucleic acid molecule may be subjected to amplification reactionsto yield an amplified product comprising the first and second targetregions and the barcode sequence or sequences complementary to thesesequences. Accordingly, the method may provide amplified productswithout the use of reverse transcription. One or more processes may beperformed within a cell bead and/or a partition, such as a droplet orwell.

Methods of Nucleic Acid Analysis

In an aspect, the present disclosure provides a method comprisingproviding a sample comprising a nucleic acid molecule (e.g., aribonucleic acid (RNA) molecule) comprising a target region and a probecomprising (i) a first probe sequence complementary to the sequence ofthe target region of the nucleic acid molecule and (ii) a second probesequence; attaching (e.g., hybridizing) the first probe sequence of theprobe to the target region of the nucleic acid molecule; providing anucleic acid barcode molecule comprising (i) a first binding sequencethat is complementary to the second probe sequence, (ii) a barcodesequence, and (iii) a second binding sequence; attaching (e.g.,hybridizing) the first binding sequence of the nucleic acid barcodemolecule to the second probe sequence of the probe; extending the probefrom an end of the second probe sequence to an end of the second bindingsequence of the nucleic acid barcode molecule to form an extendednucleic acid molecule comprising both a sequence complementary to thebarcode sequence and a sequence complementary to the target region ofthe nucleic acid molecule; denaturing the extended nucleic acid moleculefrom the nucleic acid barcode molecule and the target region of thenucleic acid molecule to regenerate the nucleic acid barcode moleculeand the nucleic acid molecule; and duplicating the extended nucleic acidmolecule. The extended nucleic acid molecule may be further amplified(e.g., using polymerase chain reactions (PCR) or linear amplification,as described herein) to facilitate the detection of the extended nucleicacid molecule or a complement thereof (e.g., an amplified product) by,e.g., sequencing.

The methods described herein may facilitate gene expression profilingwith single cell resolution using, for example, chemicalligation-mediated barcoding, amplification, and sequencing. The methodsdescribed herein may allow for gene expression analysis while avoidingthe use of specialized imaging equipment and reverse transcription,which may be highly error prone and inefficient. For example, themethods may be used to analyze a pre-determined panel of target genes ina population of single cells in a sensitive and accurate manner. In somecases, the nucleic acid molecule analyzed by the methods describedherein may be a fusion gene (e.g., a hybrid gene generated viatranslocation, interstitial deletion, or chromosomal inversion).

The nucleic acid molecule analyzed by the methods described herein maybe a single-stranded or a double-stranded nucleic acid molecule. Adouble-stranded nucleic acid molecule may be completely or partiallydenatured to provide access to a target region (e.g., a target sequence)of a strand of the nucleic acid molecule. Denaturation may be achievedby, for example, adjusting the temperature or pH of a solutioncomprising the nucleic acid molecule; using a chemical agent such asformamide, guanidine, sodium salicylate, dimethyl sulfoxide, propyleneglycol, urea, or an alkaline agent (e.g., NaOH); or using mechanicalagitation (e.g., centrifuging or vortexing a solution including thenucleic acid molecule).

The nucleic acid molecule may be an RNA molecule. The RNA molecule maybe, for example, a transfer RNA (tRNA) molecule, ribosomal RNA (rRNA)molecule, mitochondrial RNA (mtRNA) molecule, messenger RNA (mRNA)molecule, non-coding RNA molecule, synthetic RNA molecule, or anothertype of RNA molecule. For example, the RNA molecule may be an mRNAmolecule. In some cases, the nucleic acid molecule may be a viral orpathogenic RNA. In some cases, the nucleic acid molecule may be asynthetic nucleic acid molecule previously introduced into or onto acell. For example, the nucleic acid molecule may comprise a plurality ofbarcode sequences, and two or more barcode sequences may be targetregions of the nucleic acid molecule.

The nucleic acid molecule (e.g., RNA molecule) may comprise one or morefeatures selected from the group consisting of a 5′ cap structure, anuntranslated region (UTR), a 5′ triphosphate moiety, a 5′ hydroxylmoiety, a Kozak sequence, a Shine-Dalgarno sequence, a coding sequence,a codon, an intron, an exon, an open reading frame, a regulatorysequence, an enhancer sequence, a silencer sequence, a promotersequence, and a poly(A) sequence (e.g., a poly(A) tail). For example,the nucleic acid molecule may comprise one or more features selectedfrom the group consisting of a 5′ cap structure, an untranslated region(UTR), a Kozak sequence, a Shine-Dalgarno sequence, a coding sequence,and a poly(A) sequence (e.g., a poly(A) tail).

Features of the nucleic acid molecule may have any usefulcharacteristics. A 5′ cap structure may comprise one or more nucleosidemoieties joined by a linker such as a triphosphate (ppp) linker. A 5′cap structure may comprise naturally occurring nucleoside and/ornon-naturally occurring (e.g., modified) nucleosides. For example, a 5′cap structure may comprise a guanine moiety or a modified (e.g.,alkylated, reduced, or oxidized) guanine moiety such as a7-methylguanylate (m⁷G) cap. Examples of 5′ cap structures include, butare not limited to, m⁷GpppG, m⁷Gpppm⁷G, m⁷GpppA, m⁷GpppC, GpppG,m^(2,7)GpppG, m^(2,2,7)GpppG, and anti-reverse cap analogs such asm^(7,2′Ome)GpppG, m^(7,2′d)GpppG, m^(7,3′Ome)GpppG, and m^(7,3′d)GpppG.An untranslated region (UTR) may be a 5′ UTR or a 3′ UTR. A UTR mayinclude any number of nucleotides. For example, a UTR may comprise atleast 3, 5, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or morenucleotides. In some cases, a UTR may comprise fewer than 20nucleotides. In other cases, a UTR may comprise at least 100nucleotides, such as more than 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides. Similarly, a coding sequence may include any numberof nucleotides, such as at least 3, 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, or more nucleotides. A UTR, coding sequence, or other sequenceof a nucleic acid molecule may have any nucleotide or base content orarrangement. For example, a sequence of a nucleic acid molecule maycomprise any number or concentration of guanine, cytosine, uracil, andadenine bases. A nucleic acid molecule may also include non-naturallyoccurring (e.g., modified) nucleosides. A modified nucleoside maycomprise one or more modifications (e.g., alkylations, hydroxylation,oxidation, or other modification) in its nucleobase and/or sugarmoieties.

The nucleic acid molecule may comprise one or more target regions. Insome cases, a target region may correspond to a gene or a portionthereof. Each region may have the same or different sequences. Forexample, the nucleic acid molecule may comprise two target regionshaving the same sequence located at different positions along a strandof the nucleic acid molecule. Alternatively, the nucleic acid moleculemay comprise two or more target regions having different sequences.Different target regions may be interrogated by different probes. Targetregions may be located adjacent to one another or may be spatiallyseparated along a strand of the nucleic acid molecule. As used hereinwith regard to two entities, “adjacent,” may mean that the entitiesdirectly next to one other (e.g., contiguous) or in proximity to oneanother. For example, a first target region may be directly next to asecond target region (e.g., having no other entity disposed between thefirst and second target regions) or in proximity to a second targetregion (e.g., having an intervening sequence or molecule between thefirst and second target regions). In some cases, a double-strandednucleic acid molecule may comprise a target region in each strand thatmay be the same or different. For a nucleic acid molecule comprisingmultiple target regions, the methods described herein may be performedfor one or more target regions at a time. For example, a single targetregion of the multiple target regions may be analyzed (e.g., asdescribed herein) or two or more target regions may be analyzed at thesame time. Analyzing two or more target regions may involve providingtwo or more probes, where a first probe has a sequence that iscomplementary to the first target region, a second probe has a sequencethat is complementary to the second target region, etc. Each probe mayfurther comprise one or more additional sequences (e.g., additionalprobe sequences, unique molecular identifiers (UMIs), or othersequences) that are different from one another such that each probe maybind to a different nucleic acid barcode molecule. In another example,where two target regions are non-adjacent, a first target region and asecond target region may be separated by one or more gap regionsdisposed between the first target region and the second target region.

A target region of the nucleic acid molecule may have one or more usefulcharacteristics. For example, a target region may have any usefullength, base content, sequence, melting point, or other characteristic.A target region may comprise, for example, at least 10 bases, such as atleast 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, 500, or more bases. A target region may have any useful basecontent and any useful sequence and combination of bases. For example, atarget region may comprise one or more adenine, thymine, uracil,cytosine, and/or guanine bases (e.g., natural or canonical bases). Atarget region may also comprise one or more derivatives or modifiedversions of a natural or canonical base, such as an oxidized, alkylated(e.g., methylated), hydroxylated, or otherwise modified base. Similarly,a target region may comprise ribose or deoxyribose moieties andphosphate moieties or derivatives or modified versions thereof.

A target region of the nucleic acid molecule may comprise one or moresequences or features, or portions thereof, of the nucleic acidmolecule. For example, a target region may comprise all or a portion ofa UTR (e.g., a 3′ UTR or a 5′ UTR), a Kozak sequence, a Shine-Dalgarnosequence, a coding sequence, a polyA sequence, a cap structure, anintron, an exon, or any other sequence or feature of the nucleic acidmolecule.

The nucleic acid molecule (e.g., RNA molecule, such as an mRNA molecule)of a sample may be included within a cell. For example, the sample maycomprise a cell comprising the nucleic acid molecule. The cell maycomprise additional nucleic acid molecules that may be the same as ordifferent from the nucleic acid molecule of interest. In some cases, thesample may comprise a plurality of cells, and each cell may contain oneor more nucleic acid molecules. The cell may be, for example, a humancell, an animal cell, or a plant cell. In some cases, the cell may bederived from a tissue or fluid, as described herein. The cell may be aprokaryotic cell or a eukaryotic cell. The cell may be a lymphocyte suchas a B cell or T cell.

Access to a nucleic acid molecule included in a cell may be provided bylysing or permeabilizing the cell. Lysing the cell may release thenucleic acid molecule contained therein from the cell. A cell may belysed using a lysis agent such as a bioactive agent. A bioactive agentuseful for lysing a cell may be, for example, an enzyme (e.g., asdescribed herein). An enzyme used to lyse a cell may or may not becapable of carrying out additional functions such as degrading,extending, reverse transcribing, or otherwise altering a nucleic acidmolecule. Alternatively, an ionic or non-ionic surfactant such asTritonX-100, Tween 20, sarcosyl, or sodium dodecyl sulfate may be usedto lyse a cell. Cell lysis may also be achieved using a cellulardisruption method such as an electroporation or a thermal, acoustic, ormechanical disruption method. Alternatively, a cell may be permeabilizedto provide access to a nucleic acid molecule included therein.Permeabilization may involve partially or completely dissolving ordisrupting a cell membrane or a portion thereof. Permeabilization may beachieved by, for example, contacting a cell membrane with an organicsolvent (e.g., methanol) or a detergent such as Triton X-100 or NP-40.

A nucleic acid molecule or a derivative thereof (e.g., a probe-linkednucleic acid molecule, a nucleic acid molecule having one or more probeshybridized thereto, a barcoded probe-linked nucleic acid molecule, or anextended nucleic acid molecule or complement thereof) or a cellcomprising the nucleic acid molecule or a derivative thereof (e.g., acell bead) may be partitioned within a partition such as a well ordroplet, e.g., as described herein. One or more reagents may beco-partitioned with a nucleic acid molecule or a derivative thereof or acell comprising the nucleic acid molecule or a derivative thereof. Forexample, a nucleic acid molecule or a derivative thereof or a cellcomprising the nucleic acid molecule or a derivative thereof may beco-partitioned with one or more reagents selected from the groupconsisting of lysis agents or buffers, permeabilizing agents, enzymes(e.g., enzymes capable of digesting one or more RNA molecules, extendingone or more nucleic acid molecules, reverse transcribing an RNAmolecule, permeabilizing or lysing a cell, or carrying out otheractions), fluorophores, oligonucleotides, primers, probes, barcodes,nucleic acid barcode molecules (e.g., nucleic acid barcode moleculescomprising one or more barcode sequences), buffers, deoxynucleotidetriphosphates, detergents, reducing agents, chelating agents, oxidizingagents, nanoparticles, beads, and antibodies. In some cases, a nucleicacid molecule or a derivative thereof, or a cell comprising the nucleicacid molecule or a derivative thereof (e.g., a cell bead), may beco-partitioned with one or more reagents selected from the groupconsisting of temperature-sensitive enzymes, pH-sensitive enzymes,light-sensitive enzymes, reverse transcriptases, proteases, ligase,polymerases, restriction enzymes, nucleases, protease inhibitors,exonucleases, and nuclease inhibitors. For example, a nucleic acidmolecule or a derivative thereof or a cell comprising the nucleic acidmolecule or a derivative thereof may be co-partitioned with a polymeraseand nucleotide molecules. Partitioning a nucleic acid molecule or aderivative thereof or a cell comprising the nucleic acid molecule or aderivative thereof and one or more reagents may comprise flowing a firstphase comprising an aqueous fluid, the cell, and the one or morereagents and a second phase comprising a fluid that is immiscible withthe aqueous fluid toward a junction. Upon interaction of the first andsecond phases, a discrete droplet of the first phase comprising thenucleic acid molecule or a derivative thereof or a cell comprising thenucleic acid molecule or a derivative thereof (e.g., a cell bead) andthe one or more reagents may be formed. In some cases, the partition maycomprise a single cell. The cell may be lysed or permeabilized withinthe partition (e.g., droplet) to provide access to the nucleic acidmolecule of the cell.

In some embodiments, the cell may be lysed within the cell bead, and asubset of the intracellular contents may associate with the bead. Insome cases, the cell bead may comprise thioacrydite-modified nucleicacid molecules that can hybridize with nucleic acids from the cell. Forexample, a poly-T nucleic acid sequence may be thioacrydite-modified andbound to the cell bead matrix. Upon cell lysis, the cellular nucleicacids (e.g., mRNA) may hybridize with the poly-T sequence. The retainedintracellular contents may be released, for example, by addition of areducing agent, e.g, DTT, TCEP, etc. The release may occur at anyconvenient step, such as before or after partitioning.

One or more processes may be carried out within a partition. Forexample, one or more processes selected from the group consisting oflysis, permeabilization, denaturation, hybridization, extension,duplication, and amplification of one or more components of a samplecomprising the nucleic acid molecule may be performed within apartition. In some cases, multiple processes are carried out within apartition. The nucleic acid molecule or a cell comprising the nucleicacid molecule, may be co-partitioned with one or more reagents (e.g., asdescribed herein) at any useful stage of the method. For example, thenucleic acid molecule contained within a cell may be co-partitioned witha probe and one or more additional reagents prior to hybridization ofthe probe with the target region of the nucleic acid molecule.Similarly, the nucleic acid molecule or a cell comprising the nucleicacid molecule may be released from a partition at any useful stage ofthe method. For example, the nucleic acid molecule or a cell comprisingthe nucleic acid molecule may be released from the partition subsequentto hybridization of a binding sequence of a nucleic acid barcodemolecule to a sequence of a probe hybridized to the target region of thenucleic acid molecule. Alternatively, the nucleic acid molecule or acell comprising the nucleic acid molecule, and/or another component ofthe sample comprising the same, may be released from the partitionsubsequent to denaturation of a complexed extended nucleic acid moleculethat comprises a sequence complementary to the barcode sequence of anucleic acid barcode molecule and a sequence complementary to the targetregion of the nucleic acid molecule. Duplication and/or amplification ofthe extended nucleic acid molecule may then be carried out within asolution. In some cases, the solution may comprise additional extendednucleic acid molecules generated through the same process carried out indifferent partitions. Each extended nucleic acid molecule may comprise adifferent barcode sequence or a sequence complementary to a differentbarcode sequence. In this instance, the solution may be a pooled mixturecomprising the contents of two or more partitions (e.g., droplets).

Hybridization of a probe sequence of a probe to a target region of thenucleic acid molecule may be performed within or outside of a partition.In some cases, hybridization may be preceded by denaturation of adouble-stranded nucleic acid molecule to provide a single-strandednucleic acid molecule or by lysis or permeabilization of a cell. In somecases, the hybridization may occur in a cell bead comprising a cell. Thesequence of the probe that is complementary to the target region may besituated at an end of the probe. Alternatively, this sequence may bedisposed between other sequences such that when the probe sequence ishybridized to the target region, additional probe sequences extendbeyond the hybridized sequence in multiple directions. The probesequence that hybridizes to the target region of the nucleic acidmolecule may be of the same or different length as the target region.For example, the probe sequence may be shorter than the target regionand may only hybridize to a portion of the target region. Alternatively,the probe sequence may be longer than the target region and mayhybridize to the entirety of the target region and extend beyond thetarget region in one or more directions. In addition to a probe sequencecomplementary to a target region of the nucleic acid molecule, the probemay comprise one or more additional probe sequences. For example, theprobe may comprise the probe sequence complementary to the target regionand a second probe sequence. The second probe sequence may have anyuseful length and other characteristics. The probe may comprise one ormore additional sequences, such as one or more barcode sequences orunique molecule identifier (UMI) sequences. In some cases, one or moreprobe sequences of the probe may comprise a detectable moiety such as afluorophore or a fluorescent moiety.

A probe sequence of the probe may be capable of hybridizing with asequence of a nucleic acid barcode molecule. A nucleic acid barcodemolecule may comprise a first binding sequence that is complementary toa probe sequence of the probe (e.g., a second probe sequence), a barcodesequence, and a second binding sequence. A nucleic acid barcode moleculemay also comprise one or more additional functional sequences selectedfrom the group consisting of primer sequences, primer annealingsequences, and immobilization sequences. The binding sequences may haveany useful length and other characteristics. In some cases, the bindingsequence that is complementary to a probe sequence of the probe may bethe same length as the probe sequence. Alternatively, the bindingsequence may be a different length of the probe sequence. For example,the binding sequence may be shorter than the probe sequence and may onlyhybridize to a portion of the probe sequence. Alternatively, the bindingsequence may be longer than the probe sequence and may hybridize to theentirety of the probe sequence and extend beyond the probe sequence inone or more directions.

The barcode sequence of a nucleic acid barcode molecule may have anyuseful length and other characteristics (e.g., as described herein). Thenucleic acid barcode molecule may be attached to a bead such as a gelbead (e.g., as described herein). The bead may be co-partitioned withthe nucleic acid molecule or the cell comprising the nucleic acidmolecule. The bead may comprise a plurality of nucleic acid barcodemolecules that may be the same or different. The bead may comprise atleast 10,000 nucleic acid barcode molecules attached thereto. Forexample, the bead may comprise at least 100,000, 1,000,000, or10,000,000 nucleic acid barcode molecules attached thereto. In somecases, each nucleic acid barcode molecule of the plurality of nucleicacid barcode molecules may comprise a common barcode sequence. Thenucleic acid barcode molecules may further comprise an additionalbarcode sequence that may be different for each nucleic acid barcodemolecule attached to the bead. The plurality of nucleic acid barcodemolecules may be releasably attached to the bead. The plurality ofnucleic acid barcode molecules may be releasable from the bead uponapplication of a stimulus. Such a stimulus may be selected from thegroup consisting of a thermal stimulus, a photo stimulus, and a chemicalstimulus. For example, the stimulus may be a reducing agent such asdithiothreitol Application of a stimulus may result in one or more of(i) cleavage of a linkage between nucleic acid barcode molecules of theplurality of nucleic acid barcode molecules and the bead, and (ii)degradation or dissolution of the bead to release nucleic acid barcodemolecules of the plurality of nucleic acid barcode molecules from thebead. In some cases, one or more nucleic acid barcode molecules may bereleased from the bead prior to hybridization of a binding sequence of anucleic acid barcode molecule to a probe sequence of the probehybridized to the nucleic acid molecule of interest. The one or morenucleic acid barcode molecules may be released from the bead within apartition including the bead and the nucleic acid molecule (or a cellcomprising the nucleic acid molecule) and the probe. Releasing may takeplace before, after, or during hybridization of a probe sequence to atarget region of the nucleic acid molecule.

Following hybridization of a binding sequence of the nucleic acidbarcode molecule to a probe sequence of the probe hybridized to thetarget region of the nucleic acid molecule, the probe may be extendedfrom an end of the probe to an end of the nucleic acid barcode molecule.Extension may comprise the use of an enzyme (e.g., a polymerase) to addone or more nucleotides to the end of the probe. Extension may providean extended nucleic acid molecule comprising sequences complementary tothe target region of the nucleic acid molecule of interest, the barcodesequence, and one or more additional sequences of the nucleic acidbarcode molecule such as one or more binding sequences. Appropriateconditions and or chemical agents (e.g., as described herein) may thenbe applied to denature the extended nucleic acid molecule from thenucleic acid barcode molecule and the target nucleic acid molecule. Insome cases, one or more processes may involve the use of thermosensitiveagents. For example, in some cases, probes may be annealed or hybridizedunder one set of temperature conditions, and extension may occur under adifferent set of temperature conditions. In some cases, a Warm or HotStart polymerase may be used. The nucleic acid barcode molecule and thetarget nucleic acid molecule may then undergo further analysis. Forexample, a second probe that may be identical to the first probe andcomprise a probe sequence that is complementary to the target region ofthe nucleic acid molecule may hybridize to the target region, and thenucleic acid barcode molecule may hybridize to an additional probesequence of the second probe. In some cases, hybridization of thenucleic acid barcode molecule to the probe may precede hybridization ofthe probe to the target region of the nucleic acid molecule. Theextended nucleic acid molecule that has been released from the nucleicacid barcode molecule and the target nucleic acid molecule may beduplicated or amplified by, for example, one or more amplificationreactions. The amplification reactions may comprise polymerase chainreactions (PCR) and may involve the use of one or more primers orpolymerases. The extension, denaturation, and/or amplification processesmay take place within a partition. Alternatively, materials may bereleased from a partition prior to extension, denaturation, oramplification. For example, materials may be released from a partitionbetween the extension and denaturation processes. Denaturation may thentake place within a solution comprising the extended nucleic acidmolecule, nucleic acid barcode molecule, and target nucleic acidmolecule. Alternatively, materials may be released from a partitionsubsequent to denaturation and prior to amplification. In some cases,the extended nucleic acid molecule may be duplicated or amplified withina partition to provide an amplified product. The extended nucleic acidmolecule, or a complement thereof (e.g., an amplified product), may bedetected via sequencing (e.g., as described herein).

FIG. 9 schematically illustrates a representative method of analyzing anucleic acid molecule. Panel 9A shows a nucleic acid molecule 900 (e.g.,a mRNA molecule) comprising a target region 902. Probe 904 comprisesprobe sequences 906 and 908. Probe sequence 906 has a sequencecomplementary to target region 902 of nucleic acid molecule 900 andhybridizes thereto. Unhybridized probes may be optionally removed using,e.g., one or more washing steps and/or enzymatic digestion reactions.Panel 9B shows nucleic acid barcode molecule 910 comprising bindingsequence 912, adapter sequence 916 and barcode sequence 914 (whichoptionally may comprise a UMI sequence). Binding sequence 912 has asequence complementary to probe sequence 908 and hybridizes thereto.Adapter sequence 916 may comprise one or more functional sequences(e.g., a primer sequence/primer binding sequence, a sequencing primersequence (e.g., R1 or R2), a partial sequencing primer sequence (e.g.,partial R1 or partial R2), a sequence configured to attach to the flowcell of a sequencer (e.g., P5 or P7, or partial sequences thereof), abarcode sequence, UMI sequence, or complements of these sequences).Panel 9C shows extension of probe 904 (and/or barcode molecule 910) togenerate extended nucleic acid molecule 918, which comprises probesequences 906 and 908; sequence 920, which is complementary to barcodesequence 914; and sequence 922, which is complementary to adaptersequence 916. Panel 9D shows denaturation of extended nucleic acidmolecule 918 from nucleic acid molecule 900. In other embodiments, thenucleic acid extension reaction of Panel 9C generates a double strandedmolecule (comprising strand 918, e.g., similar to 924) and thedenaturation step described in Panel 9D is not performed. In still otherembodiments, nucleic acid barcode molecule 910 is a partially doublestranded molecule and is ligated to probe 904 in Panel 9C. Panel 9Eshows optional duplication or amplification of extended nucleic acidmolecule 918 (or a double stranded product comprising strand 918) togenerate amplified product 924. Amplified product 924 comprises sequence926, which is complementary to sequence 922 and the same orsubstantially the same as adapter sequence 916 of nucleic acid barcodemolecule 910; sequence 928, which is complementary to sequence 920 andthe same or substantially the same as barcode sequence 914 of nucleicacid barcode molecule 910; sequence 930, which is complementary to probesequence 908 and the same or substantially the same as binding sequence912 of nucleic acid barcode molecule 910; and sequence 932, which iscomplementary to probe sequence 906 and the same or substantially thesame as target region 902 of nucleic acid molecule 900. The barcodedproduct, or a derivative thereof, may be detected, e.g., via nucleicacid sequencing (e.g., as described herein).

In some embodiments, nucleic acid molecule 900 is present in a cell. Forinstance, in some embodiments, a cell (which is optionally fixed)comprising nucleic acid molecule 900 is permeabilized and probe 904 isadded and allowed to enter the cell and hybridize to region 902 asdescribed above. Unbound probe 904 is then washed away (and/orenzymatically digested) and the cell is lysed to release probe 904(which, in some instances, may still be hybridized to nucleic acidmolecule 900) for barcoding as described above. Alternatively, nucleicacid barcode molecule 910 is allowed to enter the permeabilized cell forbarcoding as described above.

In some embodiments, nucleic acid barcode molecule 910 is attached to abead as described elsewhere herein. For example, nucleic acid barcodemolecule 910 may be releasably attached to a bead (e.g., via labile bondas described herein). In some instances, the bead may be a gel bead asdescribed herein, e.g., a degradable gel bead. In some embodiments, apermeabilized cell comprising nucleic acid molecule 900 is incubatedwith probe 904 and the cell is then partitioned into a partition (e.g.,a droplet or well) with nucleic acid barcode molecule 910 (e.g.,attached to a bead, such as a single bead) for barcoding. In otherinstances, a cell comprising nucleic acid molecule 900, probe 904, andnucleic acid barcode molecule 910 (e.g., attached to a bead, such as asingle bead) are partitioned into a partition (e.g., a droplet or well)for probe-binding and barcoding.

In some instances, the methods described herein comprise contacting aplurality of permeabilized cells (or permeabilized nucleic or cellbeads) with one or more probes (e.g., 904) targeted to one or moreregions within one or more nucleic acid molecules (e.g., mRNAmolecules). After probe binding and removal of excess probe, theplurality of cells and a plurality of beads (e.g., gel beads) comprisingnucleic acid barcode molecules (e.g., releasably attached barcodemolecules) may then be partitioned into a plurality of partitions (e.g.,a plurality of droplets or a plurality of wells, e.g., in a microwellarray) such that at least some partitions of the plurality of partitionscomprise a single cell and a single bead. Probes (e.g., 904) may then bebarcoded as generally described in FIG. 9 . Barcoded nucleic acidmolecules may then be analyzed by any suitable technique, includingnucleic acid sequencing (e.g., Illumina sequencing).

The presently disclosed method may be applied to a single nucleic acidmolecule or a plurality of nucleic acid molecules (e.g., a plurality ofmRNA molecules). A method of analyzing a sample comprising a nucleicacid molecule may comprise providing a plurality of nucleic acidmolecules (e.g., RNA molecules, such as a cell comprising a plurality ofmRNA molecules), where each nucleic acid molecule comprises a targetregion, and a plurality of probes. In some cases, the target region ofnucleic acid molecules of the plurality of nucleic acid molecules maycomprise the same sequence. The plurality of probes may each comprise afirst probe sequence complementary to a sequence of a target region of anucleic acid molecule (e.g., mRNA molecule) of the plurality of nucleicacid molecules as well as a second probe sequence. One or more probesmay comprise the same first probe sequence. A first probe sequence of aprobe of the plurality of probes may be hybridized to a target region ofa nucleic acid molecule of the plurality of nucleic acid molecules. Abinding sequence of a nucleic acid barcode molecule of a plurality ofnucleic acid barcode molecules may hybridize to the second probesequence of a probe of the plurality of probes that is hybridized to atarget region of a nucleic acid molecule of a plurality of nucleic acidmolecules. Each nucleic acid barcode molecule of the plurality ofnucleic acid barcode molecules may comprise a barcode sequence and asecond binding sequence. The barcode sequence of each nucleic acidbarcode molecule of the plurality of nucleic acid barcode molecules maybe the same or different. Following hybridization of a binding sequenceof a nucleic acid barcode molecule of the plurality of nucleic acidbarcode molecules to a probe sequence of a probe of the plurality ofprobes that is hybridized to a target region of a nucleic acid moleculeof the plurality of nucleic acid molecules, each probe of the pluralityof hybridized probes may then be extended from an end of the probe to anend of the nucleic acid barcode molecule to which it is hybridized(e.g., an end of the second binding sequence of the nucleic acid barcodemolecule). A plurality of extended nucleic acid molecules may thereby becreated, where each extended nucleic acid molecule of the plurality ofextended nucleic acid molecules comprises a sequence complementary to atarget region of a nucleic acid molecule of the plurality of nucleicacid molecules and a sequence complementary to a barcode sequence of anucleic acid barcode molecule of the plurality of nucleic acid barcodemolecules.

In some cases, one or more processes described above may be performedwithin a partition. For example, each nucleic acid molecule of theplurality of nucleic acid molecules may be provided within a differentpartition. This may be achieved by partitioning a plurality of cellscomprising the plurality of nucleic acid molecules within a plurality ofseparate partitions, where each cell comprises a target nucleic acidmolecule and each partition of a plurality of different partitions ofthe plurality of separate partitions comprises a single cell. Access toa target nucleic acid molecule contained within a cell in a partitionmay be provided by lysing or permeabilizing the cell (e.g., as describedherein). Nucleic acid barcode molecules provided within each partitionof the plurality of different partitions of the plurality of separatepartitions may be provided attached to beads. For example, eachpartition of the plurality of different partitions of the plurality ofseparate partitions may comprise a bead comprising a plurality ofnucleic acid barcode molecules attached thereto (e.g., as describedherein). The plurality of nucleic acid barcode molecules attached toeach bead may comprise a different barcode sequence, such that eachpartition of the plurality of different partitions of the plurality ofseparate partitions comprises a different barcode sequence. Upon releaseof components from the plurality of different partitions of theplurality of separate partitions (e.g., following extension of eachprobe), each extended nucleic acid molecule may comprise a sequencecomplementary to a different barcode sequence, such that each extendednucleic acid molecule can be traced to a given partition and, in somecases, a given cell.

In another aspect, the present disclosure provides a method comprisingproviding a sample comprising a nucleic acid molecule (e.g., aribonucleic acid (RNA) molecule) having a first target region and asecond target region. The first target region may be adjacent to thesecond target region a first probe and a second probe. The first probemay comprise a first probe sequence and a second probe sequence, wherethe first probe sequence of the first probe is complementary to thefirst target region of the nucleic acid molecule. The second probe maycomprise a third probe sequence that is complementary to the secondtarget region of the nucleic acid molecule. The first probe sequence mayalso comprise a first reactive moiety, and the third probe sequence maycomprise a second reactive moiety. The sample may be subjected toconditions sufficient to hybridize (i) the first probe sequence of thefirst probe to the first target region of the nucleic acid molecule and(ii) the third probe sequence of the second probe to the second targetregion of the nucleic acid molecule such that the first reactive moietyof the first probe sequence is adjacent to the second reactive moiety ofthe third probe sequence. The reactive moieties may then be subjected toconditions sufficient to cause them to react to yield a probe-linkednucleic acid molecule comprising the first probe linked to the secondprobe. The probe-linked nucleic acid molecule may then be barcoded(e.g., within a partition) to provide a barcoded probe-linked nucleicacid molecule. Barcoding may comprise hybridizing a binding sequence ofa nucleic acid barcode molecule to the second probe sequence of thefirst probe. The first probe of the barcoded probe-linked nucleic acidmolecule may subsequently be extended from an end of the first probe toan end of the nucleic acid barcode molecule to which it is hybridized toprovide an extended nucleic acid molecule. The extended nucleic acidbarcode molecule may comprise the first probe, the second probe, asequence complementary to the barcode sequence of the nucleic acidbarcode molecule, and a sequence complementary to another sequence(e.g., another binding sequence) of the nucleic acid barcode molecule.The extended nucleic acid molecule may be denatured from the nucleicacid barcode molecule and the nucleic acid molecule of interest and thenduplicated or amplified (e.g., using polymerase chain reactions (PCR) orlinear amplification) to facilitate detection of the extended nucleicacid molecule or a complement thereof (e.g., an amplified product) by,e.g., sequencing. One or more of the methods described herein may allowfor genomic, transcriptomic, or exomic profiling with highersensitivity. One or more of the methods described herein may allow forprofiling of non-polyadenylated targets (e.g., non-poly-A RNAs), splicejunctions, single nucleotide polymorphism s (SNPs), fixed cells, etc.One or more of the methods described herein may be compatible formultiplexed analysis, such as using feature barcoding, as describedelsewhere herein.

The methods described herein may facilitate gene expression profilingwith single cell resolution using, for example, chemicalligation-mediated barcoding, amplification, and sequencing. The methodsdescribed herein may allow for gene expression analysis while avoidingthe use of enzymatic ligation, specialized imaging equipment, andreverse transcription, which may be highly error prone and inefficient.For example, the methods may be used to analyze a pre-determined panelof target genes in a population of single cells in a sensitive andaccurate manner. In some cases, the nucleic acid molecule analyzed bythe methods described herein may be a fusion gene (e.g., a hybrid genegenerated via translocation, interstitial deletion, or chromosomalinversion).

The nucleic acid molecule analyzed by the method may be asingle-stranded or double-stranded nucleic acid molecule (e.g., asdescribed herein). The nucleic acid molecule may be an RNA molecule suchas an mRNA molecule. In some cases, the nucleic acid molecule may be aviral or pathogenic RNA. In some cases, the nucleic acid molecule may bea synthetic nucleic acid molecule previously introduced into or onto acell. For example, the nucleic acid molecule may comprise a plurality ofbarcode sequences, and two or more barcode sequences may be targetregions of the nucleic acid molecule.

The nucleic acid molecule (e.g., mRNA molecule) may comprise one or morefeatures selected from the group consisting of a 5′ cap structure, anuntranslated region (UTR), a 5′ triphosphate moiety, a 5′ hydroxylmoiety, a Kozak sequence, a Shine-Dalgarno sequence, a coding sequence,a codon, an intron, an exon, an open reading frame, a regulatorysequence, an enhancer sequence, a silencer sequence, a promotersequence, and a poly(A) sequence (e.g., a poly(A) tail). Features of thenucleic acid molecule may have any useful characteristics. Additionaldetails of nucleic acid molecules are provided in the preceding section.

The nucleic acid molecule may comprise two or more target regions. Insome cases, a target region may correspond to a gene or a portionthereof. Each region may have the same or different sequences. Forexample, the nucleic acid molecule may comprise two target regionshaving the same sequence located at adjacent positions along a strand ofthe nucleic acid molecule. Alternatively, the nucleic acid molecule maycomprise two or more target regions having different sequences atadjacent positions along a strand of the nucleic acid molecule. As usedherein with regard to two entities, “adjacent,” may mean that theentities directly next to one other (e.g., contiguous) or in proximityto one another. For example, a first target region may be directly nextto a second target region (e.g., having no other entity disposed betweenthe first and second target regions) or in proximity to a second targetregion (e.g., having an intervening sequence or molecule between thefirst and second target regions). In some cases, the nucleic acidmolecule may comprise additional target regions disposed at differentlocations along the same or a different strand of the nucleic acidmolecule. For example, a double-stranded nucleic acid molecule maycomprise one or more target regions in each strand that may be the sameor different. Different target regions may be interrogated by differentprobes. For example, a first target region may be interrogated by afirst probe having a first probe sequence that is complementary to thefirst target region, and a second target region may be interrogated by asecond probe having a second probe sequence that is complementary to thesecond target region. One or both probes may further comprise one ormore additional sequences (e.g., additional probe sequences, uniquemolecular identifiers (UMIs), or other sequences). For example, thefirst probe may further comprise a second probe sequence. The secondprobe sequence of the first probe may undergo hybridization with abinding sequence of a nucleic acid barcode molecule. The second probemay also comprise an additional probe sequence. This sequence may bedifferent from the second barcode sequence of the first probe so thatthe first and second probes may hybridize to different nucleic acidbarcode molecules.

The target regions of the nucleic acid molecule may have any usefulcharacteristics (e.g., as described in the preceding section).

The nucleic acid molecule (e.g., RNA molecule, such as an mRNA molecule)of a sample may be included within a cell (e.g., as described in thepreceding section). For example, the sample may comprise a cellcomprising the nucleic acid molecule that may be, for example, a humancell, an animal cell, or a plant cell. Access to a nucleic acid moleculeincluded in a cell may be provided by lysing or permeabilizing the cell(e.g., as described in the preceding section).

Hybridization of a probe sequence of a probe to a target region of thenucleic acid molecule may be performed within or outside of a cell,partition, and/or container. In some cases, a cell may be lysed within acell bead and a subset of the intracellular contents (e.g., mRNA) may beretained in the cell bead, as described elsewhere herein. In such cases,hybridization of a probe sequence of a probe to a target region of thenucleic acid may occur prior to partitioning. In some cases,hybridization may be preceded by denaturation of a double-strandednucleic acid molecule to provide a single-stranded nucleic acid moleculeor by lysis or permeabilization of a cell. The sequence of a probe thatis complementary to a target region may be situated at an end of theprobe. Alternatively, this sequence may be disposed between othersequences such that when the probe sequence is hybridized to a targetregion, additional probe sequences extend beyond the hybridized sequencein multiple directions. A probe sequence that hybridizes to a targetregion of the nucleic acid molecule may be of the same or differentlength as the target region. For example, a probe sequence may beshorter than a target region and may only hybridize to a portion of thetarget region. Alternatively, a probe sequence may be longer than atarget region and may hybridize to the entirety of the target region andextend beyond the target region in one or more directions. In additionto a probe sequence complementary to a target region of the nucleic acidmolecule, a probe may comprise one or more additional probe sequences.For example, a probe may comprise a probe sequence complementary to atarget region and a second probe sequence. The second probe sequence mayhave any useful length and other characteristics. In an example, thefirst probe comprises a first probe sequence capable of hybridizing tothe first target region of the nucleic acid molecule of interest and asecond probe sequence, and the second probe comprises a third probesequence capable of hybridizing to the second target region of thenucleic acid molecule of interest. In some cases, the second probe mayfurther comprise a fourth binding sequence. Both the first probe and thesecond probe may comprise one or more additional sequences, such as oneor more barcode sequences or unique molecule identifier (UMI) sequences.In some cases, one or more probe sequences of a probe may comprise adetectable moiety such as a fluorophore or a fluorescent moiety.

A probe may comprise a reactive moiety. For example, a probe sequence ofa first probe capable of hybridizing to a first target region of anucleic acid molecule may comprise a first reactive moiety, and a probesequence of a second probe capable of hybridizing to a second targetregion of the nucleic acid molecule may comprise a second reactivemoiety. When the first and second probes are hybridized to the first andsecond target regions of the nucleic acid molecule, the first and secondreactive moieties may be adjacent to one another. A reactive moiety of aprobe may be selected from the non-limiting group consisting of azides,alkynes, nitrones (e.g., 1,3-nitrones), strained alkenes (e.g.,trans-cycloalkenes such as cyclooctenes or oxanorbornadiene),tetrazines, tetrazoles, iodides, thioates (e.g., phorphorothioate),acids, amines, and phosphates. For example, the first reactive moiety ofa first probe may comprise an azide moiety, and a second reactive moietyof a second probe may comprise an alkyne moiety. The first and secondreactive moieties may react to form a linking moiety. A reaction betweenthe first and second reactive moieties may be, for example, acycloaddition reaction such as a strain-promoted azide-alkynecycloaddition, a copper-catalyzed azide-alkyne cycloaddition, astrain-promoted alkyne-nitrone cycloaddition, a Diels-Alder reaction, a[3+2] cycloaddition, a [4+2] cycloaddition, or a [4+1] cycloaddition; athiol-ene reaction; a nucleophilic substation reaction; or anotherreaction. In some cases, reaction between the first and second reactivemoieties may yield a triazole moiety or an isoxazoline moiety. Areaction between the first and second reactive moieties may involvesubjecting the reactive moieties to suitable conditions such as asuitable temperature, pH, or pressure and providing one or more reagentsor catalysts for the reaction. For example, a reaction between the firstand second reactive moieties may be catalyzed by a copper catalyst, aruthenium catalyst, or a strained species such as a difluorooctyne,dibenzylcyclooctyne, or biarylazacyclooctynone. Reaction between a firstreactive moiety of a first probe sequence of a first probe hybridized toa first target region of the nucleic acid molecule and a second reactivemoiety of a third probe sequence of a second probe hybridized to asecond target region of the nucleic acid molecule may link the firstprobe and the second probe to provide a probe-linked nucleic acidmolecule. Upon linking, the first and second probes may be consideredligated. Accordingly, reaction of the first and second reactive moietiesmay comprise a chemical ligation reaction such as a copper-catalyzed 5′azide to 3′ alkyne “click” chemistry reaction to form a triazole linkagebetween two probes. In other non-limiting examples, an iodide moiety maybe chemically ligated to a phosphorothioate moiety to form aphosphorothioate bond, an acid may be ligated to an amine to form anamide bond, and/or a phosphate and amine may be ligated to form aphosphoramidate bond.

FIG. 15 illustrates examples of representative reactions. Panel 15Ashows a chemical ligation reaction of an alkyne moiety 1502 and an azidemoiety 1504 reacting under copper-mediated cycloaddition to form atriazole linkage 1506. Panel 15B shows a chemical ligation reaction of aphosphorothioate group 1508 with an iodide group 1510 to form aphosphorothioate linkage 1512. Panel 15C shows a chemical ligationreaction of an acid 1514 and amine 1516 to form an amide linkage 1518.Panel 15D shows a chemical ligation reaction of a phosphate moiety 1520and an amine moiety 1522 to form a phosphoramidate linkage 1524. Panel15E shows a conjugation reaction of two species 1526 and 1528.

In some instances, the first and second probes are hybridized to thefirst and second target regions of the nucleic acid molecule, and thefirst and second reactive moieties may be adjacent to one another. Insome cases, the probes do not comprise reactive moieties and may besubjected to a nucleic acid reaction, providing a probe-linked nucleicacid molecule. For example, the probes may be subjected to an enzymaticligation reaction, using a ligase (e.g., SplintR ligase KOD ligase,and/or T4 ligase). See, e.g., Zhang L., et al.; Archaeal RNA ligase fromthermoccocus kodakarensis for template dependent ligation RNA Biol.2017; 14(1): 36-44 for a description of KOD ligase. Following theenzymatic ligation reaction, the first and second probes may beconsidered ligated. In one embodiment, the first and second probes areboth present in a linear nucleic acid molecule. In another embodiment,the linear nucleic acid molecule is a molecular inversion probe.

In other instances, the first and second probes are hybridized to thefirst and second target regions of the nucleic acid molecule, and thefirst and second reactive moieties may not be adjacent to one another.(e.g., comprise a gap region between the first and second probes). Thefirst probe and the second probe may be positioned on (i.e., hybridizedto) the nucleic acid molecule (e.g., mRNA) one or more nucleotidesapart. For example, the first probe and the second probe may be spacedat least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000 or more nucleotides apart. In some embodiments, thenon-adjacent first and second probes may be ligated to form aprobe-linked nucleic acid molecule. The probes may be subjected to anenzymatic ligation reaction, using a ligase, e.g., SplintR ligases, T4ligases, KOD ligases, PBCV1 enzymes. Gaps between the probes may firstbe filled prior to ligation, using, for example, Mu polymerase, DNApolymerase, RNA polymerase, reverse transcriptase, VENT polymerase, Taqpolymerase, and/or any combinations, derivatives, and variants (e.g.,engineered mutants) thereof. In some embodiments, ribonucleotides areligated between the first and second probes. In some embodiments,deoxyribonucleotides are ligated between the first and second probes. Inone embodiment, the first and second probes are both present in a linearnucleic acid molecule. In another embodiment, the linear nucleic acidmolecule may form a circularized nucleic acid molecule uponhybridization to target regions. The circularized nucleic acid moleculemay then be subjected to conditions sufficient for ligation of its endsto form a circular probe-linked nucleic acid molecule.

A probe sequence of a probe (e.g., a probe of a probe-linked nucleicacid molecule) may be capable of hybridizing with a sequence (e.g.,binding sequence) of a nucleic acid barcode molecule. In other cases, aprobe may comprise a barcode molecule. A nucleic acid barcode moleculemay comprise a first binding sequence that is complementary to a probesequence of a probe (e.g., a second probe sequence), a barcode sequence,and a second binding sequence. In some cases, the binding sequence of aprobe, a barcode nucleic acid molecule, or both, may be known and maybind to a target of interest (e.g., mRNA encoding a gene of interest).In some cases, the binding sequence may be degenerate (i.e., randomlygenerated). Employing degenerate or known sequences may be used in wholetranscriptome or exome analysis or for targeted RNA sequencing,respectively. A nucleic acid barcode molecule may also comprise one ormore additional functional sequences selected from the group consistingof primer sequences, primer annealing sequences, and immobilizationsequences. The binding sequences may have any useful length and othercharacteristics. In some cases, the binding sequence that iscomplementary to a probe sequence of a probe may be the same length asthe probe sequence. Alternatively, the binding sequence may be adifferent length of the probe sequence. For example, the bindingsequence may be shorter than the probe sequence and may only hybridizeto a portion of the probe sequence. Alternatively, the binding sequencemay be longer than the probe sequence and may hybridize to the entiretyof the probe sequence and extend beyond the probe sequence in one ormore directions.

In some cases, the barcode nucleic acid molecule may hybridize to abinding sequence of one or more probes or adapters in a specificorientation. In some embodiments, a barcode may be configured to bind tothe 3′ end of a probe, an adapter, or an adapter-ligated probe. In someinstances, binding of a probe to a barcode molecule is direct (e.g.,through direct hybridization) or indirect, e.g., using a splint sequenceas described elsewhere herein (e.g., FIG. 20 ). In some instances,probes and/or barcode molecules may comprise one or more ribonucleotidesto facilitate binding and ligation. In one non-limiting example, abinding sequence of a probe may comprise a pair of 3′ terminalribonucleotides. A barcode nucleic acid molecule may be phosphorylatedat the 5′ end and may associate with the ribonucleotides via a splintmolecule. The barcode nucleic acid molecule may then be ligated to the3′ end of the probe. Hybridization and ligation of a barcode nucleicacid molecule at the 3′ end of a probe may be advantageous as thisprocess may minimize downstream amplification artifacts, minimizebarcode exchange, and may be compatible with removal of unligatedprobes.

In some cases, a first probe with a first probe sequence capable ofhybridizing with a first target region of the nucleic acid molecule maycomprise a second probe sequence capable of hybridizing with a sequenceof a nucleic acid barcode molecule, and a second probe capable ofhybridizing with a second target region of the nucleic acid molecule maynot comprise a sequence capable of hybridizing with a nucleic acidbarcode molecule. In other cases, the second probe may also comprise aprobe sequence capable of hybridizing with a sequence of a nucleic acidbarcode molecule. The first nucleic acid barcode molecule to which afirst probe hybridizes may be different from a second nucleic acidbarcode molecule to which a second probe hybridizes. For example, thefirst and second nucleic acid barcode molecules may comprise one or moredifferent binding sequences and/or different barcode sequences.

In some cases, a first probe with a first probe sequence capable ofhybridizing with a first target region of the nucleic acid molecule maycomprise a second probe sequence capable of hybridizing with a firstsequence of a nucleic acid adaptor molecule. The nucleic acid adaptormolecule may comprise this first sequence, or a complement thereof, anda second sequence that can hybridize with a first sequence of a nucleicacid barcode molecule. The nucleic acid adaptor molecule may alsocomprise a third sequence such as a primer region for downstream PCR(e.g., sequencing primer sequence), a barcode sequence, etc. The nucleicacid adaptor molecule may have any combination and derivatives orvariants of the abovementioned sequences. In one non-limiting example,the nucleic acid adaptor molecule may comprise a first sequence thatenables hybridization of the nucleic acid adapter molecule to the firstprobe and a second sequence that enables hybridization of the nucleicacid adapter molecule to a nucleic acid barcode molecule. The nucleicacid barcode molecule may hybridize to the adapter molecule. In someembodiments, the nucleic acid barcode molecule can comprise additionalfunctional sequences, such as a barcode sequence, sequencing primersequence, a UMI, a spacer sequence, and a plurality of ribonucleotides.

In some embodiments, the barcode nucleic acid molecule may comprise asplint nucleic acid sequence. The barcode nucleic acid molecule may bepartially double-stranded and comprise a binding sequence and a barcodesequence. In some cases, the binding sequence may be complementary to aportion of the first probe, the second probe, or both probes.Hybridization of the binding sequence to the first probe or second probeor both probes may occur in a partition or outside of a partition. Thenucleic acid barcode molecule may then be ligated to the first probe,the second probe, or both, using, for example, chemical or enzymaticligation.

The barcode sequence of a nucleic acid barcode molecule may have anyuseful length and other characteristics (e.g., as described herein). Thenucleic acid barcode molecule may be attached to a bead such as a gelbead (e.g., as described herein). The bead may be co-partitioned withthe nucleic acid molecule or the cell comprising the nucleic acidmolecule. The bead may comprise a plurality of nucleic acid barcodemolecules that may be the same or different. The bead may comprise atleast 10,000 nucleic acid barcode molecules attached thereto. Forexample, the bead may comprise at least 100,000, 1,000,000, or10,000,000 nucleic acid barcode molecules attached thereto. In somecases, each nucleic acid barcode molecule of the plurality of nucleicacid barcode molecules may comprise a common barcode sequence. Thenucleic acid barcode molecules may further comprise an additionalbarcode sequence that may be different for each nucleic acid barcodemolecule attached to the bead. The plurality of nucleic acid barcodemolecules may be releasably attached to the bead. The plurality ofnucleic acid barcode molecules may be releasable from the bead uponapplication of a stimulus. Such a stimulus may be selected from thegroup consisting of a thermal stimulus, a photo stimulus, and a chemicalstimulus. For example, the stimulus may be a reducing agent such asdithiothreitol. Application of a stimulus may result in one or more of(i) cleavage of a linkage between nucleic acid barcode molecules of theplurality of nucleic acid barcode molecules and the bead, and (ii)degradation or dissolution of the bead to release nucleic acid barcodemolecules of the plurality of nucleic acid barcode molecules from thebead. In some cases, one or more nucleic acid barcode molecules may bereleased from the bead prior to hybridization of a binding sequence of anucleic acid barcode molecule to a probe sequence of the probehybridized to the nucleic acid molecule of interest. The one or morenucleic acid barcode molecules may be released from the bead within apartition including the bead and the nucleic acid molecule (or a cellcomprising the nucleic acid molecule) and the probe. Releasing may takeplace before, after, or during hybridization of a probe sequence to atarget region of the nucleic acid molecule.

FIG. 10 schematically illustrates a representative method of analyzing anucleic acid molecule. Panel 10A shows a nucleic acid molecule 1000(e.g., a mRNA molecule) comprising target regions 1002 and 1004. In someinstances, target regions 1002 and 1004 are adjacent to one another.Probe 1006 comprises probe sequence 1008, binding sequence 1010, andreactive moiety 1012. Probe 1014 comprises probe sequence 1016 andreactive moiety 1018. Probe sequence 1008 of probe 1006 is complementaryto target region 1002 of nucleic acid molecule 1000. Similarly, probesequence 1016 of probe 1014 is complementary to target region 1004 ofnucleic acid molecule 1000. Panel 10B shows probe sequence 1008 of probe1006 hybridized to target region 1002 and probe sequence 1016 of probe1014 hybridized to target region 1004. In some instances, reactivemoiety 1012 of probe 1006 and reactive moiety 1018 of probe 1014 areadjacent to one another. Panel 10C shows linking moiety 1020 producedthrough a reaction of reactive moieties 1012 and 1018. In some cases,moieties 1012 and 1018 are ligated chemically (e.g., click chemistry),and in other cases, enzymatically (e.g., a ligase, such as SplintR, KODligase, or T4 ligase). Linked probes 1006 and 1014 comprise aprobe-linked nucleic acid molecule comprising sequences 1010, 1008, and1016. Panel 10D shows nucleic acid barcode molecule 1022 comprisingadapter sequence 1028, barcode sequence 1026 (which optionally maycomprise a UMI sequence), and binding sequence 1024, which iscomplementary to binding sequence 1010. Adapter sequence 1028 maycomprise one or more functional sequences (e.g., a primersequence/primer binding sequence, a sequencing primer sequence (e.g., R1or R2), a partial sequencing primer sequence (e.g., partial R1 orpartial R2), a sequence configured to attach to the flow cell of asequencer (e.g., P5 or P7, or partial sequences thereof), a barcodesequence, UMI sequence, or complements of these sequences). Nucleic acidbarcode molecule 1022 is then hybridized to binding sequence 1010 of theprobe-linked nucleic acid molecule. A barcoded probe-linked nucleic acidmolecule is then generated using, e.g., a nucleic acid extensionreaction and/or ligation reaction as described in, e.g., Panel 9C. Insome cases, probe 1014 may comprise an additional binding sequence (notshown). Probe sequence 1016 may hybridize to another nucleic acidbarcode molecule or primer comprising a sequence complementary to probesequence 1016. In some cases, moieties 1012 and 1018 may not be reactiveand can be ligated using an enzyme (e.g., a ligase, such as SplintR, T4ligase, KOD ligase, etc.). In some instances, where target regions 1002and 1004 are not adjacent to one another, probe 1006 and/or 1014 may beextended in a nucleic acid extension reaction and ligated together asdescribed elsewhere herein.

In some instances, following hybridization of a binding sequence 1024 ofthe nucleic acid barcode molecule 1022 to a binding sequence 1010 of aprobe (e.g., probe-linked nucleic acid molecule) hybridized to a targetregion of the nucleic acid molecule 1000, the probe may be extended in anucleic acid extension reaction to generate a barcoded probe-linkednucleic acid molecule. Extension may comprise the use of an enzyme(e.g., a polymerase) to add one or more nucleotides to the end of theprobe and/or nucleic acid barcode molecule. Extension may provide abarcoded probe-linked nucleic acid molecule comprising sequencescomplementary to: (i) the first 1002 and second 1004 target regions ofthe nucleic acid molecule of interest 1000, (ii) the barcode sequence1026, and (iii) one or more additional sequences of the nucleic acidbarcode molecule such as one or more adapter sequences (e.g., 1028). Insome instances, the barcoded probe-linked nucleic acid molecule issingle stranded. In other instances, the barcoded probe-linked nucleicacid molecule is double stranded. In some instances, where the barcodedprobe-linked nucleic acid molecule is single stranded, appropriateconditions and or chemical agents (e.g., as described herein) may thenbe applied to denature the extended nucleic acid molecule from thetarget nucleic acid molecule. The target nucleic acid molecule may thenundergo further analysis. For example, another set of probes mayhybridize to the target regions of the nucleic acid molecule, and anucleic acid barcode molecule may be appended to a probe sequence of oneof the additional probes. In some cases, hybridization of the nucleicacid barcode molecule to the first probe may precede hybridization ofthe first and second probes to the target region of the nucleic acidmolecule. The barcoded probe-linked nucleic acid molecule may beduplicated or amplified by, for example, one or more amplificationreactions, which may in some instances be isothermal. The amplificationreactions may comprise polymerase chain reactions (PCR) and may involvethe use of one or more primers or polymerases. The one or more primersmay comprise one or more functional sequences (e.g., a primersequence/primer binding sequence, a sequencing primer sequence (e.g., R1or R2), a partial sequencing primer sequence (e.g., partial R1 orpartial R2), a sequence configured to attach to the flow cell of asequencer (e.g., P5 or P7, or partial sequences thereof), etc.) and mayfacilitate addition of said one or more functional sequences to theextended nucleic acid molecule. The barcoded probe-linked nucleic acidmolecule, or a derivative thereof, may be detected via nucleic acidsequencing (e.g., as described herein).

In some embodiments, nucleic acid molecule 1000 is present in a cell.For instance, in some embodiments, a cell (which is optionally fixed)comprising nucleic acid molecule 1000 is permeabilized and probes 1006and 1014 are added and allowed to enter the cell and hybridize toregions 1002 and 1004 as described above. Unbound probes are then washedaway (and/or enzymatically digested) and the probes enzymatically orchemically linked together as described elsewhere herein. The cell maythen be lysed to release probe-linked nucleic acid molecule 1030 (which,in some instances, may still be hybridized to nucleic acid molecule1000) for barcoding as described above. Alternatively, nucleic acidbarcode molecule 1022 is allowed to enter the permeabilized cell forbarcoding as described above. In some embodiments, nucleic acid barcodemolecule 1022 is attached to a bead as described elsewhere herein. Forexample, nucleic acid barcode molecule 1022 may be releasably attachedto a bead (e.g., via labile bond as described herein). In someinstances, the bead may be a gel bead as described herein, e.g., adegradable gel bead. In some embodiments, a permeabilized cellcomprising nucleic acid molecule 1000 is incubated with probes 1006 and1014 and the cell is then partitioned into a partition (e.g., a dropletor well) with nucleic acid barcode molecule 1022 (e.g., attached to abead, such as a single bead) for barcoding. In other instances, a cellcomprising nucleic acid molecule 1000, probes 1006 and 1014, and nucleicacid barcode molecule 1022 (e.g., attached to a bead, such as a singlebead) are partitioned into a partition (e.g., a droplet or well) forprobe-binding and barcoding.

In some instances, the methods described herein comprise contacting aplurality of permeabilized cells (or permeabilized nucleic or cellbeads) with one or more probes (e.g., probes 1006 and 1014) targeted toone or more regions (e.g., 1002 and 1004) within one or more nucleicacid molecules (e.g., mRNA molecules). After probe binding and removalof excess probe, the plurality of cells and a plurality of beads (e.g.,gel beads) comprising nucleic acid barcode molecules (e.g., releasablyattached barcode molecules) may then be partitioned into a plurality ofpartitions (e.g., a plurality of droplets or a plurality of wells, e.g.,in a microwell array) such that at least some partitions of theplurality of partitions comprise a single cell and a single bead. Probesmay then be barcoded as generally described above. Barcoded nucleic acidmolecules or derivatives thereof may then be optionally furtherprocessed and analyzed by any suitable technique, including nucleic acidsequencing (e.g., Illumina sequencing).

FIG. 12 schematically illustrates a representative method of analyzing anucleic acid molecule. Panel 12A shows a nucleic acid molecule 1200(e.g., a mRNA molecule) comprising target regions 1202 and 1204. In someinstances, target regions 1202 and 1204 are adjacent to one another.Probe 1206 comprises probe sequence 1208, binding sequence 1210 andreactive moiety 1212. Probe 1214 comprises probe sequences 1216, adaptersequence 1248, and reactive moiety 1218. Probe sequence 1208 of probe1206 is complementary to target region 1202. Similarly, probe sequence1216 of probe 1214 is complementary to target region 1204. Panel 12Bshows probe sequence 1208 of probe 1206 hybridized to target region 1202and probe sequence 1216 of probe 1214 hybridized to target region 1204.In some instances, reactive moiety 1212 of probe 1206 and reactivemoiety 1218 of probe 1214 are adjacent to one another.

Panel 12C shows linking moiety 1220 produced through a reaction ofreactive moieties 1212 and 1218. In some cases, moieties 1212 and 1218are ligated chemically (e.g., click chemistry), and in other cases,enzymatically (e.g., a ligase, such as SplintR, KOD ligase, or T4ligase). Linked probes 1206 and 1214 comprise a probe-linked nucleicacid molecule 1230 comprising sequences 1210, 1208, 1216, and 1248.Panel 12D shows nucleic acid barcode molecule 1222 comprising bindingsequence 1224, barcode sequence 1226 (which optionally may comprise aUMI sequence), and binding sequence 1228, which is complementary tobinding sequence 1210. Adapter sequence 1228 may comprise one or morefunctional sequences (e.g., a primer sequence/primer binding sequence, asequencing primer sequence (e.g., R1 or R2), a partial sequencing primersequence (e.g., partial R1 or partial R2), a sequence configured toattach to the flow cell of a sequencer (e.g., P5 or P7, or partialsequences thereof), a barcode sequence, UMI sequence, or complements ofthese sequences). Nucleic acid barcode molecule 1222 is then hybridizedto binding sequence 1210 of the probe-linked nucleic acid molecule 1230.A barcoded probe-linked nucleic acid molecule 1240 is then generatedusing, e.g., a nucleic acid extension reaction and/or ligation reactionas described previously (see, e.g., Panel 9C). The barcoded probe-linkednucleic acid molecule 1240 may comprise sequences 1248, 1216, 1208,1210, 1232 (complementary to barcode sequence 1226) and 1234(complementary to adapter sequence 1228). In some instances, thebarcoded probe-linked nucleic acid molecule 1240 is single stranded(e.g., only 1230 or 1222 is extended). In other instances, the barcodedprobe-linked nucleic acid molecule 1240 is double stranded (e.g., both1230 and 1222 are extended). In some instances, where the barcodedprobe-linked nucleic acid molecule 1240 is single stranded, appropriateconditions and or chemical agents (e.g., as described herein) may thenbe applied to denature the extended nucleic acid molecule from thetarget nucleic acid molecule. The barcoded probe-linked nucleic acidmolecule 1240 may be duplicated or amplified by, for example, one ormore amplification reactions, which may in some instances be isothermal.The amplification reactions may comprise polymerase chain reactions(PCR) and may involve the use of one or more primers or polymerases. Theone or more primers may comprise one or more functional sequences (e.g.,a primer sequence/primer binding sequence, a sequencing primer sequence(e.g., R1 or R2), a partial sequencing primer sequence (e.g., partial R1or partial R2), a sequence configured to attach to the flow cell of asequencer (e.g., P5 or P7, or partial sequences thereof), etc.) and mayfacilitate addition of said one or more functional sequences to theextended nucleic acid molecule. The barcoded probe-linked nucleic acidmolecule 1240, or a derivative thereof, may be detected via nucleic acidsequencing (e.g., as described herein).

In some embodiments, nucleic acid molecule 1200 is present in a cell.For instance, in some embodiments, a cell (which is optionally fixed)comprising nucleic acid molecule 1200 is permeabilized and probes 1206and 1214 are added and allowed to enter the cell and hybridize toregions 1202 and 1204 as described above. Unbound probes are then washedaway (and/or enzymatically digested) and the probes enzymatically orchemically linked together as described elsewhere herein. The cell maythen be lysed to release probe-linked nucleic acid molecule 1230 (which,in some instances, may still be hybridized to nucleic acid molecule1200) for barcoding as described above. Alternatively, nucleic acidbarcode molecule 1222 is allowed to enter the permeabilized cell forbarcoding as described above. In some embodiments, nucleic acid barcodemolecule 1222 is attached to a bead as described elsewhere herein. Forexample, nucleic acid barcode molecule 1222 may be releasably attachedto a bead (e.g., via labile bond as described herein). In someinstances, the bead may be a gel bead as described herein, e.g., adegradable gel bead. In some embodiments, a permeabilized cellcomprising nucleic acid molecule 1200 is incubated with probes 1206 and1214 and the cell is then partitioned into a partition (e.g., a dropletor well) with nucleic acid barcode molecule 1222 (e.g., attached to abead, such as a single bead) for barcoding. In other instances, a cellcomprising nucleic acid molecule 1200, probes 1206 and 1214, and nucleicacid barcode molecule 1222 (e.g., attached to a bead, such as a singlebead) are partitioned into a partition (e.g., a droplet or well) forprobe-binding and barcoding. Nucleic acid barcode molecules and probesmay be designed in any suitable 5′ to 3′ configuration. For example, anucleic acid barcode molecule attached to a bead may be attached to thebead at the 3′ end of the nucleic acid barcode molecule or at the 5′ endof the nucleic acid barcode molecule.

In some instances, the methods described herein comprise contacting aplurality of permeabilized cells (or permeabilized nucleic or cellbeads) with one or more probes (e.g., probes 1206 and 1214) targeted toone or more regions (e.g., 1202 and 1204) within one or more nucleicacid molecules (e.g., mRNA molecules). After probe binding and removalof excess probe, the plurality of cells and a plurality of beads (e.g.,gel beads) comprising nucleic acid barcode molecules (e.g., releasablyattached barcode molecules) may then be partitioned into a plurality ofpartitions (e.g., a plurality of droplets or a plurality of wells, e.g.,in a microwell array) such that at least some partitions of theplurality of partitions comprise a single cell and a single bead. Probes(e.g., 1230) may then be barcoded as generally described above. Barcodednucleic acid molecules (e.g., 1240) or derivatives thereof may then beoptionally further processed and analyzed by any suitable technique,including nucleic acid sequencing (e.g., Illumina sequencing).

In some cases, a nucleic acid barcode molecule (e.g., 1222) may belinked to the probe-linked nucleic acid molecule (e.g., 1230) via anadapter molecule. FIG. 20 schematically illustrates a representativemethod of analyzing a nucleic acid molecule using such adaptermolecules. Panel 20A shows a probe-linked nucleic acid molecule, such asthose described in, e.g., FIG. 10 , and FIG. 12 (e.g., 1230). Panel 20Bshows splint molecule 2021, which comprises a binding sequence 2022complementary to a sequence of an adapter (e.g., 908, 1010, 1210, etc.)in a probe-linked nucleic acid molecule (e.g., 1230). The splintmolecule 2021 may also comprise a binding sequence 2023. In someembodiments, the binding sequence 2023 may comprise or moreribonucleotides, such as ribo-guanines or ribo-cytosines. In someinstances, the one or more ribonucleotides are present at the end (e.g.,5′ terminus or 3′ terminus) of the adapter sequence. In some instances,the splint molecule 2021 is a single stranded, or a partially doublestranded molecule. Panel 20C shows hybridization of a barcode nucleicacid molecule 2022 to splint molecule 2021. The barcode nucleic acidmolecule 2022 comprises an adapter sequence 2028, barcode sequence 2026,and binding sequence 2024, which is complementary to binding sequence2023 of splint molecule 2021. Adapter sequence 2028 may comprise one ormore functional sequences (e.g., a primer sequence/primer bindingsequence, a sequencing primer sequence (e.g., R1 or R2), a partialsequencing primer sequence (e.g., partial R1 or partial R2), a sequenceconfigured to attach to the flow cell of a sequencer (e.g., P5 or P7, orpartial sequences thereof), a barcode sequence, UMI sequence, orcomplements of these sequences). In some cases, the binding sequence2024 of the barcode nucleic acid molecule 2022 comprises a plurality ofribonucleotides, such as ribo-cytosines or ribo-guanines. In someinstances, the one or more ribonucleotides are present at the end (e.g.,5′ terminus or 3′ terminus) of the barcode nucleic acid molecule 2022.Following hybridization of the barcode nucleic acid molecule 2022,ligation (e.g., chemically or enzymatically) of the splinted,probe-linked nucleic acid molecule and barcode molecule 2022 may occur,to form, e.g., barcoded nucleic acid molecule 2040 as shown in Panel20D. The barcoded probe-linked nucleic acid molecule 2040 may comprisesequences 1248, 1216, 1208, 1210, 2024 (complementary to bindingsequence 2023), 2025 (complementary to barcode sequence 2026) and 2029(complementary to adapter sequence 2028). Alternatively, the splinted,probe-linked nucleic acid molecule hybridized to the nucleic acidbarcode molecule may be barcoded using a nucleic acid extension reactionas previously described. In some embodiments, a splint is not utilized,but instead the nucleic acid barcode molecule is partially doublestranded and comprises a single stranded portion comprising, e.g.,sequence 2022 to facilitate hybridization to the probe linked molecule1230. In some instances, the barcoded probe-linked nucleic acid moleculeis single stranded. In other instances, the barcoded probe-linkednucleic acid molecule is double stranded. The extended nucleic acidmolecule may subsequently be subjected to one or more amplificationreactions and/or further processing, such as those described in, e.g.,FIG. 12 . Splint molecule 2021 may be a DNA molecule or may be an RNAmolecule.

In some instances, splint molecule 2021 is pre-hybridized to the barcodenucleic acid molecule 2022 to form a splint nucleic acid molecule. Thesplint nucleic acid molecule may be used in, e.g., Panel 20C tohybridize to the probe-linked nucleic acid molecule.

FIG. 21 schematically illustrates a representative method of analyzing anucleic acid molecule using first and second probe molecules, an adaptermolecule, and a barcode nucleic acid molecule. Panel 21A shows a nucleicacid molecule 2100 comprising adjacent target regions 2102 and 2104.Nucleic acid molecule 2100 is an mRNA molecule comprising a polyAsequence at its 3′ end. Probe 2006 comprises probe sequences 2108 and2110 and probe 2114 comprises probe sequences 2116 and 2148 and loopsequence 2147. Probe sequence 2108 of probe 2006 is complementary totarget region 2102 and comprises reactive moiety 2112. Similarly, probesequence 2116 of probe 2114 is complementary to target region 2104 andcomprises reactive moiety 2118. Panel 21B shows probe sequence 2108 ofprobe 2006 hybridized to target region 2102 and probe sequence 2116 ofprobe 2114 hybridized to target region 2104. Reactive moiety 2112 ofprobe 2006 and reactive moiety 2118 of probe 2114 are adjacent to oneanother. An adapter molecule 2121 may also be introduced with probes2106 and 2114. Panel 21C shows hybridization of an adapter molecule 2121and barcode molecule 2122. The adapter molecule 2121 comprises a bindingsequence that may hybridize with probe sequence 2110 of probe 2106. Theadapter molecule 2121 may also comprise a spacer sequence 2023. In someembodiments, the spacer sequence 2023 may comprise a plurality ofribonucleotides, such as ribo-guanines or ribo-cytosines. Panel 21Dshows hybridization of a barcode nucleic acid molecule 2122 to theadapter molecule 2121. The barcode nucleic acid molecule 2122 comprisesa primer sequence 2128 (e.g., sequencing primer sequence), barcodesequence 2126, and binding sequence 2124, which is complementary to thespacer sequence 2023 of adapter molecule 2121. In some cases, thebinding sequence 2124 of the barcode nucleic acid molecule 2122comprises a plurality of ribonucleotides, such as ribo-cytosines orribo-guanines. Panel 21D illustrates digestion of excess probemolecules. An exonuclease (e.g., a 3′ exonuclease) 2130 may optionallybe used to digest unhybridized probe molecules 2106, 2114 and adaptermolecules 2121. Panel 21E shows ligation of the barcode molecule and theprobes. Linking moiety 2132 may be produced through a reaction ofreactive moieties 2112 and 2118. In some cases, moieties 2112 and 2118are ligated using click chemistry, and in other cases, an enzyme (e.g.,SplintR, KOD ligase, T4 ligase) may be used. Ligation of the probemolecules can produce a probe-linked molecule. Similarly, the barcodemolecule 2122 may be linked by a linking moiety 2132 to one of theprobes or the probe-linked molecule, generating a barcoded, probe-linkedmolecule. Further, extension of the linked probes of the probe-linkednucleic acid molecule may occur, to form an extended nucleic acidmolecule similar to that shown in FIG. 12 .

As will be appreciated, one or more processes described herein may occurinside a partition (e.g., well or droplet) or outside a partition (e.g.,in bulk). One or more processes may occur in any convenient or usefulorder. For example, in some embodiments, a first probe may be hybridizedto the target nucleic acid molecule. The first probe may then bebarcoded, e.g., using an adapter molecule and a barcode molecule, asplinted barcode molecule, or any combination or derivatives thereof.The barcode molecule and the probe may be ligated (e.g., using clickchemistry or enzymatically). In some cases, the unhybridized probes maythen be digested (e.g., using an exonuclease). Subsequently, a secondprobe molecule may be introduced, which may hybridize to the targetnucleic acid molecule, adjacent to the barcoded probe molecule. Thesecond probe molecule may then be ligated (e.g. using click chemistry orenzymatically) to form a barcoded probe-linked nucleic acid molecule. Insome cases, the barcoding may occur prior to, during, or followingpartitioning. Similarly, ligation and/or digestion may occur in apartition or outside of a partition.

FIG. 16 schematically illustrates a method of ligating non-adjacentprobes to form a probe-linked nucleic acid molecule. Panel 16A shows anucleic acid molecule 1600 comprising non-adjacent target regions 1602and 1604. Nucleic acid molecule 1600 is an mRNA molecule comprising apolyA sequence at its 3′ end. Probe 1606 comprises probe sequences 1608and 1610 and probe 1614 comprises probe sequences 1616 and moiety 1618.Probe sequence 1608 of probe 1606 is complementary to target region1602. Similarly, probe sequence 1616 of probe 1614 is complementary totarget region 1604 and comprises a moiety 1618 onto which a polymerasemay bind. Panel 16B shows probe sequence 1608 of probe 1606 hybridizedto target region 1602 and probe sequence 1616 of probe 1614 hybridizedto target region 1604. A polymerase 1620, such as Mu polymerase or DNApolymerase, extends probe 1616 by adding complementary ribonucleotides(e.g., ribonucleoside tri-phosphate (rNTP)) or deoxyribonucleotides(e.g., deoxyribonucleotide triphosphate (dNTP)), respectively (agap-fill reaction). Panel 16C shows probes 1606 and extended probe 1614as adjacent to one another. Panel 16D shows a ligation reaction of probe1606 and extended probe 1614. Ligation may occur enzymatically, forexample, by using a T4RNA ligase, KOD ligase, or a PBCV1 ligase, to forma probe-linked nucleic acid molecule 1622. Downstream analysis maysubsequently be performed, such as barcoding and amplification, similarto as shown in Panels 12 D-F in FIG. 12 .

FIG. 17 schematically shows an alternative method barcoding nucleic acidprobes using adaptor nucleic acid molecules. Panel 17A shows a nucleicacid molecule 1700 comprising a target region 1702. Nucleic acidmolecule 1700 is an mRNA molecule comprising a polyA sequence at its 3′end. Probe 1706 comprises probe sequences 1708 and adaptor sequences1710. Probe sequence 1708 of probe 1706 is complementary to targetregion 1702. Panel 17B shows probe sequence 1708 of probe 1706hybridized to target region 1702. An adaptor nucleic acid molecule 1712comprises a sequence 1714 that hybridizes with the adaptor sequence 1710of the nucleic acid probe 1706, and modular sequences 1716, 1718.Modular sequences 1716, 1718 may comprise, for example, a PCR primersequence, a barcode, a constant sequence, and/or any variants orderivatives thereof. Panel 17C schematically shows a method of barcodingthe probe nucleic acid 1706. A barcode nucleic acid molecule 1720comprises a hybridization sequence 1722 that hybridizes with the adaptornucleic acid molecule 1712 and a barcode sequence 1724. Hybridization ofthe barcode nucleic acid molecule may occur prior to or duringpartitioning. Following hybridization, other nucleic acid reactions maybe performed, such as extension using DNA polymerase, to generatedouble-stranded, barcoded, nucleic acid probes (not shown). Subsequentamplification and sequencing may be performed. While FIGS. 10-12, 13,20, 21 depict the first probe and the second probe as adjacent, it willbe appreciated that these are for illustrative purposes only and are notmeant to be limiting. In certain embodiments, the first probe and thesecond probe may not be adjacent, as depicted in FIG. 16 . Thus, any ofthe processes, components, reagents, variations and derivatives of FIGS.10-12, 13, 20, 21 , may also apply to probes that are non-adjacent.Similarly, any of the processes, components, reagents, variations, andderivatives of FIG. 16 may also be applicable to those schemes depictedin FIGS. 10-12, 13, 20, 21 .

In some cases, probe molecules that attach to the same target nucleicacid molecule may be linked to one another. For example, a single probemolecule (e.g., a probe nucleic acid molecule) may comprise (i) a firstprobe moiety at a first end that comprises a sequence complementary to afirst target region of a nucleic acid molecule and (ii) a second probemoiety at a second end that comprises a sequence complementary to asecond target region of the nucleic acid molecule that is adjacent tothe first target region. A single probe molecule may comprise additionalsequences, such as a sequencing primer binding site, or a primer sitefor downstream processing, e.g., rolling circle amplification. In someembodiments, the first probe and/or the second probe may comprise acleavable linker. In some cases, the cleavable linker may comprise arestriction site and may be cleaved upon addition of a biologicalstimulus (e.g., restriction enzyme). In some embodiments, the cleavablelinker may be cleaved upon the addition of a stimulus, e.g., a chemical,thermal, or photo stimulus. Upon hybridization of the first and secondprobe moieties to the target nucleic acid molecule, the first and secondprobe moieties may be adjacent and the probe molecule and target nucleicacid molecule may form a circular nucleic acid product. The circularnucleic acid product may then be subjected to conditions sufficient forligation of the nucleic acid product, forming a circular probe-linkednucleic acid molecule. In some embodiments, the probe-linked nucleicacid molecule may be circularized. In some cases, linking of probes mayoccur before circularization or alternatively, linking of probes mayoccur simultaneously or subsequently to circularization. In someembodiments, circularization may occur via a splint nucleic acid, suchas a circularization nucleic acid molecule. In such an embodiment, acircularization nucleic acid molecule may hybridize to a sequence on thefirst probe and a sequence on the second probe to form a circularnucleic acid product. In some embodiments, the first and second probemoieties may be connected as a single probe moiety. In some embodiments,the single probe moiety may be a circular nucleic acid product. In someembodiments, the single probe moiety may comprise single-strandedsequences that may be connected via a splint nucleic acid, such as acircularization nucleic acid molecule.

Hybridization kinetics of a circular nucleic acid product may besubstantially different from those of a corresponding linear productinvolving two disconnected probes. In some cases, the use of a singleprobe molecule comprising two probe moieties may result in enhancedsensitivity of a target region of a nucleic acid molecule. For example,the use of a single probe molecule comprising two probe moieties mayresult in an increased number of target nucleic acid molecules havingtwo probe moieties attached thereto relative to the use of twodisconnected probes. Circularization of nucleic acid moieties may alsofacilitate removal of unwanted nucleic acid species and unhybridizedprobes by permitting the use of exonucleases without affecting ligationproducts. In some cases, unwanted nucleic acid species and unhybridizedprobes may be removed from a solution or partition including a circularnucleic acid product subsequent to its formation. For example, acircular nucleic acid product may be formed in a solution, and unwantedand unhybridized materials removed from the solution prior to barcodingor other processing. In such an example, the circular nucleic acidproduct may then be partitioned with one of more materials including oneor more nucleic acid barcode molecules (e.g., coupled to a bead, asdescribed herein) or nucleic acid binding molecules to undergo furtherprocessing. Alternatively, a circular nucleic acid product may be formedwithin a partition and hybridize with a nucleic acid barcode moleculeand/or nucleic acid binding molecule within the partition to generate abarcoded circular nucleic acid product. The barcoded circular nucleicacid product may then be released from the partition to undergo furtherprocessing. A circular nucleic acid product may be opened at any usefultime. For example, the circular nucleic acid product may be openfollowing removal of unwanted and unhybridized materials. Alternatively,the circular nucleic acid product may be opened subsequent tohybridization of a nucleic acid barcode molecule and/or nucleic acidbinding molecule to the circular nucleic acid product to generate abarcoded circular nucleic acid product. In some embodiments, thecircular nucleic acid product may comprise a labile or cleavable linker.For example, the circular nucleic acid product may comprise arestriction site that is recognized by one or more restriction enzymes.Addition of one or more restriction enzymes may open the nucleic acidproduct. In another example, the circular nucleic acid product maycomprise a photo- or thermal-sensitive linker that may be cleaved uponaddition of light or heat. In some cases, a circular nucleic acidproduct may be amplified by rolling circle amplification (RCA) prior orsubsequent to partitioning of the circular nucleic acid product. The useof RCA may increase efficiency of a barcoding process by generatingmultiple targets from the same original ligation event. An RCA productmay be less susceptible to loss prior to partitioning due to its largesize. An RCA product may be digested within a partition prior to abarcoding process by hybridization of a complementary probe and arestriction enzyme or other targeted endonuclease. RCA may be used incombination with or as an alternative to PCR.

In some embodiments, a first probe or a second probe may comprise asequence that allows for further processing. In some cases, the firstprobe or the second probe may comprise a site. In some cases, the firstprobe and the second probe may be connected (e.g., the first probe andthe second probe are parts of the same probe) and may comprise atransposition site. In some cases, the first probe and the second probemay form a circular nucleic acid product that comprises a transpositionsite. In some embodiments, a transposase may be used to add sequences tothe first probe or the second probe or the circular nucleic acidproduct. For example, a transposase may be loaded with a transposaseloop sequence. The transposase loop sequence may comprise sequences thatmay be used for further processing. For example, the transposase loopsequence may comprise a primer sequencing site, a barcode sequence, asequencing primer sequence, a restriction site, a UMI sequence, a spacersequence, an adapter sequence, and any combinations, variations, orderivatives thereof. In some cases, the transposase may introduce thetransposase loop sequence into the first probe, the second probe, or thecircular nucleic acid molecule. In some cases, the transposase may alsointroduce nicks or gaps in the first probe, the second probe, or thecircular nucleic acid molecule. In such cases, the nicks or gaps may befilled, e.g., using one or more enzymes (e.g., polymerase, ligase).Further processing, e.g., amplification, rolling circle amplificationmay generate double-stranded probe molecules. In some cases, thedouble-stranded probe molecules may comprise a restriction site sequenceand a barcode sequence and may be cleaved, e.g., upon addition of arestriction enzyme, to generate barcoded nucleic acid fragments. Furtherprocessing may be performed, such as an amplification reaction, togenerate a sequencing library.

A transposase generally refers to an enzyme that is configured to bind anucleic acid molecule, cleave the nucleic acid molecule and insert anucleic acid sequence into the nucleic acid molecule (and optionallyfragment the molecule, e.g., a tagmentation reaction). In some cases, atransposase can be configured to bind to a specific site on the nucleicacid molecule. In some cases, a transposase can be configured to bind toa random site on the nucleic acid molecule. Moreover, in some cases, atransposase can be configured to bind and optionally fragment openchromatin (e.g., euchromatin). Non-limiting examples of transposasesinclude: a Tn transposase (e.g., Tn3, Tn5, Tn7, Tn10, Tn552, Tn903), aMuA tranposase, a Vibhar transposase (e.g., from Vibrio harveyi), aprokaryotic transposase, any member of the hAT superfamily oftransposases (e.g., Hermes), Ac-Ds, Ascot-1, Bs1, Cin4, Copia, En/Spm, Felement, hobo, Hsmar1, Hsmar2, IN (HIV), IS1, IS2, IS3, IS4, IS5, IS6,IS10, IS21, IS30, IS50, IS51, IS150, IS256, IS407, IS427, IS630, IS903,IS911, IS982, IS1031, ISL2, L1, Mariner, P element, Tam3, Tc1, Tc3, Tel,THE-1, Tn/O, TnA, Tol1, Tol2, TnlO, and Tyl. In some cases, thetransposase may be derived from any of the above, such as a transposaseincluding one or more mutations or modifications. In certain instances,a transposase related to and/or derived from a parent transposase cancomprise a peptide fragment with at least about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% amino acid sequence homology to acorresponding peptide fragment of the parent transposase. The peptidefragment can be at least about 10, about 15, about 20, about 25, about30, about 35, about 40, about 45, about 50, about 60, about 70, about80, about 90, about 100, about 150, about 200, about 250, about 300,about 400, or about 500 amino acids in length. For example, atransposase derived from Tn5 can comprise a peptide fragment that is 50amino acids in length and about 80% homologous to a correspondingfragment in a parent Tn5 transposase. Action of a transposase (e.g.,insertion) may be facilitated and/or triggered by addition of one ormore cations, such as one or more divalent cations (e.g., Ca²⁺, Mg²⁺, orMn²⁺) In a particular aspect, the transposase is a hyperactivetransposase, such as Tn5.

FIGS. 13A-B schematically illustrates a representative example ofnucleic acid molecule analysis. Panel 13A of FIG. 13A shows probemolecule 1305 (e.g., a molecular inversion probe) comprising probemoiety 1306 at a first end and probe moiety 1314 at a second end. Probemoiety 1306 has a sequence complementary to target region 1302 ofnucleic acid molecule 1300 (e.g., an mRNA molecule), while probe moiety1314 has a sequence complementary to target region 1304 of nucleic acidmolecule 1300. Probe moiety 1306 may comprise reactive moiety 1312, andprobe moiety 1314 may comprise reactive moiety 1318. When probe moieties1306 and 1314 are hybridized to nucleic acid molecule 1300, reactivemoieties 1312 and 1318 may be adjacent. Probe moieties 1306 and 1314 arelinked by a linking sequence. In some instances, the linking sequencecomprises adapter sequence 1322, cleavable moiety 1323, and bindingsequence 1324. Adapter sequence 1322 may comprise one or more functionalsequences (e.g., a primer sequence/primer binding sequence, a sequencingprimer sequence (e.g., R1 or R2), a partial sequencing primer sequence(e.g., partial R1 or partial R2), a sequence configured to attach to theflow cell of a sequencer (e.g., P5 or P7, or partial sequences thereof),a barcode sequence, UMI sequence, or complements of these sequences).The linking sequence may also comprise one or more nucleic acidsequences and/or other moieties (amino acids, peptides, proteins, PEGmoieties, hydrocarbon chains, or other linkers). In some embodiments,the linking sequence may comprise cleavable moiety 1323, such as amoiety comprising a thermolabile, photocleavable, or enzymaticallycleavable bond. When probe moieties 1306 and 1314 are hybridized tonucleic acid molecule 1300, reactive moieties 1312 and 1318 may beadjacent.

Panel 13B of FIG. 13A shows ligation (e.g., chemical ligation, such asusing a click chemistry reaction, or enzymatic ligation such as using aligase) of reactive moieties 1312 and 1318 to form a linking moiety1320, thereby circularizing probe 1305. As described elsewhere herein,linking moiety 1320 may comprise a triazole moiety generated by reactionof an alkyne moiety and an azide moiety. The ligation reaction ofreactive moieties 1312 and 1318 may involve the use of a catalyst suchas a copper species or a strained alkene and may take place within oroutside of a partition. In some embodiments, the circular nucleic acidproduct may be cleaved and linearized by addition of a stimulus, e.g.,biological, chemical, thermal or photo-stimulus. In one non-limitingexample, the linking sequence may comprise a restriction site andapplication of a restriction enzyme cleaves site 1323, therebylinearizing probe 1305. In some instances, prior to barcoding,circularized probe 1305 is subjected to rolling circle amplification togenerate multiple copies of probe sequence 1305. The concatemer of 1305can be resolved to molecules suitable for barcoding by, e.g., cleavingcleavable moiety 1323. In some instances, cleavable moiety 1323 is arestriction site and the rolling circle amplification product can becleaved by digesting the concatemer with a restriction enzyme specificof the restriction site. In some embodiments, adapter sequence 1322comprises a UMI such that digested products from rolling circleamplification will each comprise a UMI to identify the probe 1305 oforigin.

Panel 13C of FIG. 13A shows hybridization of sequence 1335 of nucleicacid barcode molecule 1332 to binding sequence 1324. Followinghybridization, linearized probe 1305 (which may or may not have beensubjected to rolling circle amplification and digestion) may be barcodedby, e.g., nucleic acid extension and/or ligation as previously describedherein (e.g., FIG. 9 , FIG. 10 , FIG. 12 , etc.). The barcoding reactionmay be facilitated through use of a splint molecule as describedelsewhere herein (e.g., FIG. 20 ).

Panel 13A of FIG. 13B shows probe molecule 1310 and probe molecule 1340bound to nucleic acid molecule 1300. Probe molecule 1310 comprises aprobe sequence 1306, adapter sequence 1322, cleavable moiety 1323 (e.g.,as described above), and reactive moiety 1312. Probe sequence 1306 iscomplementary to target region 1302 of nucleic acid molecule 1300 (e.g.,a mRNA molecule). Probe molecule 1340 comprises probe sequence 1314,binding sequence 1324, and reactive moiety 1318. Probe sequence 1314 iscomplementary to target region 1304 of nucleic acid molecule 1300 (e.g.,a mRNA molecule).

Probe molecules 1310 and 1340 may also comprise one or more additionalnucleic acid sequences and/or other moieties (amino acids, peptides,proteins, PEG moieties, hydrocarbon chains, or other linkers). Acircularization nucleic acid molecule 1328 may be used to connect probemolecules 1310 and 1340. The circularization nucleic acid molecule 1328may comprise sequences 1330 and 1332. Sequence 1330 of thecircularization nucleic acid molecule may be capable of hybridizing witha sequence of probe molecule 1310, and sequence 1332 of thecircularization nucleic acid molecule may be capable of hybridizing witha sequence (e.g., 1324) of probe molecule 1340. After hybridization ofthe circularization nucleic acid molecule with probe molecules 1310 and1340, the two molecules may be ligated together at 1321. The ligationmay be chemical or enzymatic as described elsewhere herein. When probemoieties 1306 and 1314 are hybridized to nucleic acid molecule 1300,reactive moieties 1312 and 1318 may be adjacent. Probe moieties 1306 and1314 are linked by a linking sequence 1330. The ligation of 1306 to 1314may be chemical or enzymatic as described elsewhere herein. As describedelsewhere herein, the linking moiety (e.g., 1320 or 1321) may comprise atriazole moiety generated by reaction of an alkyne moiety and an azidemoiety. The ligation reaction of reactive moieties 1312 and 1318 mayinvolve the use of a catalyst such as a copper species or a strainedalkene and may take place within or outside of a partition. In somecases, moieties 1312 and 1318 may be adjacent and may not comprisereactive moieties. In such cases, moieties 1312 and 1318 may be ligatedenzymatically (e.g., using a ligase). In some instances, 1310 is ligatedto 1340 at 1321 prior to ligation at 1320. In some instances, 1310 isligated to 1340 at 1320 prior to ligation at 1321. In some instances,1310 is ligated to 1340 at 1321 and 1320 simultaneously or substantiallysimultaneously. The circularized molecule 1350 may be barcoded asdescribed in previously in FIG. 13A. Barcoded molecules or derivativesthereof may then be analyzed by, e.g., nucleic acid sequencing.

One or more processes of the presently disclosed method may be carriedout within a partition (e.g., as described herein). For example, one ormore processes selected from the group consisting of lysis,permeabilization, denaturation, hybridization, extension, duplication,and amplification of one or more components of a sample comprising thenucleic acid molecule may be performed within a partition. In somecases, multiple processes are carried out within a partition.

The nucleic acid molecule or a derivative thereof (e.g., a probe-linkednucleic acid molecule, a nucleic acid molecule having one or more probeshybridized thereto, a barcoded probe-linked nucleic acid molecule, or anextended nucleic acid molecule or complement thereof) or a cellcomprising the nucleic acid molecule or a derivative thereof (e.g., acell bead), as well as additional components (e.g., probes, nucleic acidbarcode molecules, and reagents), may be provided within a partition. Insome cases, the probes may be hybridized to the target regions of thenucleic acid molecule and linked or ligated to one another inside apartition. Alternatively, the probes may be hybridized to the targetregions of the nucleic acid molecule and linked or ligated to oneanother outside of a partition. For example, the nucleic acid moleculeor a cell comprising the nucleic acid molecule may be provided in acontainer other than a partition and undergo hybridization of the probeswithin the initial container or another container that is not apartition. In some cases, a cell may be permeabilized (e.g., asdescribed herein) to provide access to the nucleic acid molecule ofinterest therein and hybridization of the probes to the target regionsof the nucleic acid molecule of interest may take place within the cell.Ligation of the probes hybridized to the target regions of the nucleicacid molecule may then be initiated (e.g., under suitable conditions andthrough introduction of an appropriate catalyst) to provide aprobe-linked nucleic acid molecule. For example, reaction between afirst probe comprising an azide moiety and a second probe comprising analkyne moiety may be catalyzed by a copper catalyst. Excess probes andcatalyst may then be washed away and the cell may be partitioned (e.g.,as described herein) for further analysis and processing. In anotherexample, ligation of the hybridized probes may take place within apartition. Extension, denaturation, and/or amplification processes mayalso take place within a partition.

The nucleic acid molecule or a derivative thereof (e.g., a probe-linkednucleic acid molecule, a nucleic acid molecule having one or more probeshybridized thereto, a barcoded probe-linked nucleic acid molecule, or anextended nucleic acid molecule or complement thereof) or the cellcomprising the nucleic acid molecule or a derivative thereof (e.g., acell bead) may be co-partitioned with one or more reagents (e.g., asdescribed herein) at any useful stage of the method. For example, thenucleic acid molecule or a derivative thereof contained within a cellmay be co-partitioned with one or more reagents following generation ofthe probe-linked nucleic acid molecule. Similarly, the nucleic acidmolecule or a derivative thereof or a cell comprising the nucleic acidmolecule or a derivative thereof may be released from a partition at anyuseful stage of the method. For example, the nucleic acid molecule or aderivative thereof or a cell comprising the nucleic acid molecule or aderivative thereof may be released from the partition subsequent tohybridization of a binding sequence of a nucleic acid barcode moleculeto a probe-linked nucleic acid molecule (e.g., to a sequence of a probehybridized to the target region of the nucleic acid molecule) to providea barcoded probe-linked nucleic acid molecule. In another example,release from the partition may take place subsequent to extension of thebarcoded probe-linked nucleic acid molecule to provide an extendednucleic acid molecule that comprises a sequence complementary to thebarcode sequence of a nucleic acid barcode molecule and one or moresequences complementary to one or more target regions of the nucleicacid molecule. Alternatively, the nucleic acid molecule or a derivativethereof or a cell comprising the nucleic acid molecule or a derivativethereof may be released from a partition subsequent to denaturation ofan extended nucleic acid molecule from the nucleic acid molecule and thenucleic acid barcode molecule. Duplication and/or amplification of theextended nucleic acid molecule may then be carried out within asolution. In some cases, such a solution may comprise additionalextended nucleic acid molecules and/or complements thereof generatedthrough the same process carried out in different partitions. Eachextended nucleic acid molecule or complement thereof (e.g., amplifiedproduct) may comprise a different barcode sequence or a sequencecomplementary to a different barcode sequence. In this instance, thesolution may be a pooled mixture comprising the contents of two or morepartitions (e.g., droplets).

One or more additional components such as one or more reagents may beco-partitioned with a nucleic acid molecule or derivative thereof or acell comprising a nucleic acid molecule or a derivative thereof (e.g.,as described in the preceding section).

In some cases, the methods described herein may be used to facilitategene expression analysis. For example, a target nucleic acid moleculecomprising a hybrid gene may be contacted by a plurality of differentprobes. One or more probes of the plurality of probes may have asequence complementary to a first portion of the hybrid gene (e.g., afirst target region), and one or more probes of the plurality of probesmay have a sequence complementary to a second portion of the hybrid gene(e.g., a second target region) in proximity to the first portion of thehybrid gene. The two probes may each comprise a reactive moiety suchthat, upon hybridization to the hybrid gene and exposure to appropriatereaction conditions, the two probes may ligate to one another. Thesolution including the probe-ligated hybrid gene may undergo processingto remove unhybridized probes and may be partitioned with one or morereagents including one or more nucleic acid barcode molecules. A nucleicacid barcode molecule included within the partition including theprobe-ligated hybrid gene may have a sequence complementary to asequence of a probe hybridized to the hybrid gene and may hybridizethereto to generate a barcoded probe-ligated hybrid gene. Subsequentextension and amplification may take place within or outside of thepartition. Following amplification to generate an amplified productcomprising sequences of portions of the hybrid gene, or complementsthereof, the amplified product may be detected using sequencing.Resultant sequence reads may be used to determine the components of thehybrid gene.

The presently disclosed method may be applied to a single nucleic acidmolecule or a plurality of nucleic acid molecules. A method of analyzinga sample comprising a nucleic acid molecule may comprise providing aplurality of nucleic acid molecules (e.g., RNA molecules), where eachnucleic acid molecule comprises a first target region and a secondtarget region, a plurality of first probes, and a plurality of secondprobes. In some cases, one or more target regions of nucleic acidmolecules of the plurality of nucleic acid molecules may comprise thesame sequence. The first and second target regions of a nucleic acidmolecule of the plurality of nucleic acid molecules may be adjacent toone another. The plurality of first probes may each comprise a firstprobe sequence complementary to the sequence of a first target region ofa nucleic acid molecule of the plurality of nucleic acid molecules aswell as a second probe sequence. A first probe sequence of a first probeof the plurality of first probes may comprise a first reactive moiety.One or more first probes of the plurality of first probes may comprisethe same first probe sequence and/or the same second probe sequence. Theplurality of second probes may each comprise a third probe sequencecomplementary to the sequence of a second target region of a nucleicacid molecule of the plurality of nucleic acid molecules. The pluralityof second probes may further comprise a fourth probe sequence. A thirdprobe sequence of a second probe of the plurality of second probes maycomprise a second reactive moiety. One or more probes of the secondprobes of the plurality of second probes may comprise the same thirdprobe sequence and/or, if present, the same fourth probe sequence. Afirst probe sequence of a first probe of the plurality of first probesmay hybridize to first target region of a nucleic acid molecule of theplurality of nucleic acid molecules. A third probe sequence of a secondprobe of the plurality of second probes may hybridize to the secondtarget region of a nucleic acid molecule of the plurality of nucleicacid molecules. The first and third probe sequences hybridized to thefirst and second target regions, respectively, of a nucleic acidmolecule of the plurality of nucleic acid molecules may be adjacent toone another such that a first reactive moiety of the first probesequence is adjacent to a second reactive moiety of the third probesequence. The first and second reactive moieties of the first and secondprobes hybridized to nucleic acid molecules of the plurality of nucleicacid molecules may react to provide a plurality of probe-linked nucleicacid molecules. A binding sequence of a nucleic acid barcode molecule ofa plurality of nucleic acid barcode molecules may hybridize to thesecond probe sequence of a first probe of the plurality of first probesthat is hybridized to a first target region of a nucleic acid moleculeof a plurality of nucleic acid molecules or a probe-linked nucleic acidmolecule of the plurality of probe-linked nucleic acid molecules. Eachnucleic acid barcode molecule of the plurality of nucleic acid barcodemolecules may comprise a barcode sequence and a second binding sequence.The barcode sequence of each nucleic acid barcode molecule of theplurality of nucleic acid barcode molecules may be the same ordifferent. Following hybridization of a binding sequence of a nucleicacid barcode molecule of the plurality of nucleic acid barcode moleculesto a second probe sequence of a first probe of the plurality of firstprobes that is hybridized to a first target region of a nucleic acidmolecule of the plurality of nucleic acid molecules or a probe-linkednucleic acid molecule of the plurality of probe-linked nucleic acidmolecules, each first probe of the plurality of hybridized probes maythen be extended from an end of the probe to an end of the nucleic acidbarcode molecule to which it is hybridized (e.g., an end of the secondbinding sequence of the nucleic acid barcode molecule). A plurality ofextended nucleic acid molecules may thereby be created, where eachextended nucleic acid molecule of the plurality of extended nucleic acidmolecules comprises a sequence complementary to the first target regionof a nucleic acid molecule of the plurality of nucleic acid molecules, asequence complementary to the second target region of a nucleic acidmolecule of the plurality of nucleic acid molecules, a second probesequence of a first probe of the plurality of first probes, a sequencecomplementary to a barcode sequence of a nucleic acid barcode moleculeof the plurality of nucleic acid barcode molecules, and one or moresequences complementary to one or more additional sequences (e.g.,binding or barcode sequences) of a nucleic acid barcode molecule of theplurality of nucleic acid barcode molecules.

In some cases, one or more processes described above may be performedwithin a partition. For example, each nucleic acid molecule of theplurality of nucleic acid molecules may be provided within a differentpartition. This may be achieved by partitioning a plurality of cellscomprising the plurality of nucleic acid molecules within a plurality ofseparate partitions, where each cell comprises a target nucleic acidmolecule and each partition of a plurality of different partitions ofthe plurality of separate partitions comprises a single cell. Theplurality of cells may be partitioned prior or subsequent tohybridization of probes to target regions of the nucleic acid moleculesof interest included therein and linking of the probes to provideprobe-linked nucleic acid molecules. Access to a target nucleic acidmolecule or derivative thereof (e.g., as described herein) containedwithin a cell in a partition may be provided by lysing or permeabilizingthe cell (e.g., as described herein). Nucleic acid barcode moleculesprovided within each partition of the plurality of different partitionsof the plurality of separate partitions may be provided attached tobeads. For example, each partition of the plurality of differentpartitions of the plurality of separate partitions may comprise a beadcomprising a plurality of nucleic acid barcode molecules attachedthereto (e.g., as described herein). The plurality of nucleic acidbarcode molecules attached to each bead may comprise a different barcodesequence, such that each partition of the plurality of differentpartitions of the plurality of separate partitions comprises a differentbarcode sequence. Upon release of components from the plurality ofdifferent partitions of the plurality of separate partitions (e.g.,following extension of each probe), each extended nucleic acid moleculemay comprise a sequence complementary to a different barcode sequence,such that each extended nucleic acid molecule can be traced to a givenpartition and, in some cases, a given cell.

FIG. 14 illustrates a sample workflow for a method of analyzing aplurality of nucleic acid molecules comprising chemical-ligationmediated amplification. Nucleic acid molecules 1404, 1406, and 1408 areprovided within container 1402. Each nucleic acid molecule comprises afirst target region and a second target region indicated by dashedlines. The first target regions of each nucleic acid molecule may be thesame or different. Similarly, the second target regions of each nucleicacid molecule may be the same or different. A plurality of first probes1403 and a plurality of second probes 1405 may be provided in container1402. First probes of the plurality of first probes 1403 may comprise afirst probe sequence that is complementary to the first target region ofnucleic acid molecule 1404, 1406, and/or 1408 and a second probesequence. First probe sequences of the plurality of first probes 1403may comprise a first reactive moiety. Second probes of the plurality ofsecond probes 1405 may comprise a third probe sequence that iscomplementary to the second target region of nucleic acid molecule 1404,1406, and/or 1408. Third probe sequences of the plurality of secondprobes 1405 may comprise a second reactive moiety. A first probesequence of first probes of the plurality of first probes 1403 mayhybridize to the first target regions of nucleic acid molecules 1404,1406, and 1408. Similarly, a second probe sequence of second probes ofthe plurality of second probes 1405 may hybridize to the second targetregions of nucleic acid molecules 1404, 1406, and 1408. The first andsecond reactive moieties of the first and third probe sequences may thenreact to provide probe-linked nucleic acid molecules 1411, 1413, and1415.

In process 1410, probe-linked nucleic acid molecules 1411, 1413, and1415 may be co-partitioned with beads 1418, 1420, and 1422 into separatedroplets 1412, 1414, and 1416 such that each droplet includes a singleprobe-linked nucleic acid molecule and a single bead. Each bead maycomprise a plurality of nucleic acid barcode molecules attached thereto.Bead 1418 comprises nucleic acid barcode molecule 1424, bead 1420comprises nucleic acid barcode molecule 1426, and bead 1422 comprisesnucleic acid barcode molecule 1428. Nucleic acid barcode molecules 1424,1426, and 1428 each comprise first and second binding sequences and abarcode sequence. The barcode sequences of nucleic acid barcodemolecules 1424, 1426, and 1428 are different such that each dropletcomprises a different barcode sequence.

In process 1430, nucleic acid barcode molecules 1424, 1426, and 1428 arereleased from their respective beads (e.g., by application of a stimulusthat degrades or dissolves the bead) within their respective droplets. Abinding sequence of nucleic acid barcode molecules 1424, 1426, and 1428hybridizes to the second probe sequence of probe-linked nucleic acidmolecules 1411, 1413, and 1415, respectively, to provide a barcodedprobe-linked nucleic acid molecule within each droplet. The barcodedprobe-linked nucleic acid molecule within each droplet then undergoesextension to provide complexed extended nucleic acid molecules 1432,1434, and 1436 comprising extended nucleic acid molecules 1433, 1435,and 1437. Extended nucleic acid molecules 1433, 1435, and 1437 comprisesequences complementary to a barcode sequence and the sequences of thetarget regions of the nucleic acid molecule from which they derive. Forexample, extended nucleic acid molecule 1433 comprises sequencescomplementary to the sequences of the target regions of nucleic acidmolecule 1404 and a sequence complementary to the barcode sequence ofnucleic acid barcode molecule 1424.

In process 1438, the contents of droplets 1412, 1414, and 1416 arepooled to provide a pooled mixture 1440 comprising complexed extendednucleic acid molecules 1432, 1434, and 1436. Complexed extended nucleicacid molecules 1432, 1434, and 1436 may then be denatured from thenucleic acid molecule and nucleic acid barcode molecule to which theyare hybridized to provide extended nucleic acid molecules 1433, 1435,and 1437. Extended nucleic acid molecules 1433, 1435, and 1437 may thenbe amplified to provide amplified products corresponding to eachextended nucleic acid molecule. The amplified products will comprisesequences that are the same or substantially the same as the barcodesequence and sequences of the target regions of the nucleic acidmolecule from which they derive. For example, the amplified productcorresponding to extended nucleic acid molecule 1433 comprises sequencesthat are the same or substantially the same as the sequences of thetarget regions of nucleic acid molecule 1404 and a sequence that is thesame or substantially the same as the barcode sequence of nucleic acidbarcode molecule 1424. Because each extended nucleic acid molecule andeach amplified product comprises a different barcode sequence orcomplement thereof, the extended nucleic acid molecules and amplifiedproducts can be traced back to particular nucleic acid molecules and, insome cases, to particular cells. This barcoding method may thereforefacilitate rapid analysis of nucleic acid molecules through, forexample, sequencing without the need for reverse transcription.

In one aspect, the present invention provides methods of analysis thattarget specific sequences (e.g., RNA sequences) with a molecularinversion probe. In one embodiment, the molecular inversion probe canform a circularized nucleic acid molecule upon hybridization to targetspecific sequences.

FIG. 18 illustrates an example workflow for a method of analyzing aplurality of nucleic acid molecules comprising enzymaticligation-mediated amplification. 1800 is a fixed and permeabilized cellcomprising nucleic acid molecules 1802. Each nucleic acid molecule 1802comprises a first target region and a second target region. The firsttarget regions of each nucleic acid molecule may be the same ordifferent. Similarly, the second target regions of each nucleic acidmolecule may be the same or different. The first and second targetregions of each nucleic acid molecule may be adjacent to one another. Aplurality of first probes 1804 comprising first and second probesequences that hybridize with the first and second target regions,respectively, may be introduced into the cell 1800. The probes 1804 maybe provided as linear molecules and may comprise adapter sequences suchas a PCR primer region, a sequencing site primer region, and/or a spacerregion, as described elsewhere herein. The first probe sequence of theplurality of probes 1804 may hybridize to the first target regions ofnucleic acid molecules 1802. Upon hybridization of the probes to thetarget regions, a circularized nucleic acid molecule may be formed.Similarly, the second probe sequence of the plurality of probes 1804 mayhybridize to the second target regions of nucleic acid molecules 1802.In some cases, the first probe sequence and the second target probesequence are adjacent to each other. In some cases, they arenon-adjacent and may be ligated using polymerases, e.g., Mu polymerase,as described elsewhere herein. In some cases, the first and second probesequences of probes 1804 comprise reactive moieties. Followinghybridization, excess, unhybridized probes may be removed via a washstep 1805. The first and second probe sequences may then be connectedvia introduction of enzymes (e.g., polymerases, ligases) or through achemical reaction (e.g., click chemistry of reactive moieties),generating a probe-linked nucleic acid molecule 1806.

In process 1808, probe-linked nucleic acid molecules 1806 within cell1800 may be co-partitioned with barcode nucleic acid molecules 1810. Thebarcode nucleic acid molecules may comprise adaptor regions including,but not limited to, a unique molecular identifier sequence, a PCR primersequence, a spacer sequence, and sequencing site primer region. Thebarcode nucleic acid molecules may be attached to beads (not shown).Each bead may comprise a plurality of nucleic acid barcode moleculesattached thereto. A binding sequence of nucleic acid barcode molecule1810 hybridizes to a sequence of the probe 1804 of the probe-linkednucleic acid molecules 1806, to provide a barcoded probe-linked nucleicacid molecule 1812. The barcoded probe-linked nucleic acid molecule 1812then undergoes a nucleic acid reaction 1813 such as amplification, e.g.,Phi29-based rolling circle amplification, to provide barcoded ampliconsof interest 1814, which comprise sequences complementary to thesequences of the target regions of nucleic acid molecule 1802, asequence complementary to the barcode sequence of nucleic acid barcodemolecule 1810, and any adaptor sequences of probe 1804.

In process 1816, the contents of the one or more partitions are pooled.Barcoded amplicons of interest 1814 may then be subjected to conditionssufficient for library preparation. In some cases, the barcodedamplicons of interest may be subjected to nucleic acid reactions, suchas amplification (e.g., PCR). The amplified products will comprisesequences that are the same or substantially the same as the barcodesequence and sequences of the target regions of the nucleic acidmolecule from which they derive. The amplified products can be tracedback to particular nucleic acid molecules and, in some cases, toparticular cells. This barcoding method may therefore facilitate rapidanalysis of nucleic acid molecules through, for example, sequencingwithout the need for reverse transcription.

FIG. 19 illustrates an example workflow for a method of analyzing aplurality of nucleic acid molecules comprising chemicalligation-mediated amplification of nucleic acids in cell beads. 1900 isa cell bead comprising dissolvable nucleic acid molecule capturemoieties 1901. These moieties may be thioacrydite-conjugated nucleicacid molecules that are bound to the gel bead matrix. Within the cellbead are nucleic acid molecules 1902, which comprise a target region. Aplurality of first probes 1904 comprising a probe sequence thathybridizes with the target region, respectively, may be introduced intothe cell bead 1900. The probes 1904 may additionally comprise adaptersequences such as a PCR primer region, a sequencing site primer region,and/or a spacer region, as described elsewhere herein. The probes 1904may also comprise a reactive moiety 1903. Following hybridization,excess, unhybridized probes may be removed via a wash step 1905.

In process 1908, the cell bead 1900 comprising nucleic acid molecules1902 is co-partitioned with barcode nucleic acid molecules 1910 whichcomprise a reactive moiety. The partition comprises conditionssufficient to release the nucleic acid molecules 1902 from the cell beadmatrix. In some cases, a reducing agent such as DTT may be used torelease the nucleic acid molecules from the cell bead into thepartition. The barcode nucleic acid molecules may be attached to beads(not shown). Each bead may comprise a plurality of nucleic acid barcodemolecules attached thereto. The partition may comprise conditionssufficient to release the nucleic acid barcode molecules from the beadsinto the partition. The barcode nucleic acid molecule 1910 may associatewith the probe 1904 that is hybridized to the nucleic acid molecule1902. The barcode nucleic acid molecule 1910 and the probe 1904 may thenbe ligated, e.g., via click chemistry of the reactive moieties on thebarcode nucleic acid molecule and the reactive moiety on the probe 1904,to provide a barcoded, probe-linked nucleic acid molecule 1912. Reactionyield may be enhanced, for example, by incorporating splint nucleic acidsequences that hybridize with the spacer adapter sequences. For example,the barcode nucleic acid molecule 1910 may comprise a sequence (e.g.,overhang sequence, not shown) that may hybridize with an adaptersequence (e.g., spacer sequence) on the probe 1904. Followinghybridization, the reactive moieties on the barcode nucleic acidmolecule 1910 and the reactive moiety on the probe 1904 may be ligatedto provide a barcoded, probe-linked nucleic acid molecule. In othernon-limiting examples, the barcode nucleic acid molecule 1910 may bepartially double-stranded and comprise a sequence (e.g., overhangsequence) to form a splint nucleic acid sequence that can partiallyhybridize with the probe 1904 and be ligated to provide a barcoded,probe-linked nucleic acid molecule that is partially double-stranded.

In process 1916, the contents of the one or more partitions are pooled.The barcoded probe-linked nucleic acid molecules 1912 may then besubjected to conditions sufficient for library preparation. In somecases, the barcoded probe-linked nucleic acid molecules are cleaned up.In a non-limiting example of cleanup, samples may be enriched orpurified via a magnetic-based pulldown assay of the of nucleic acidmolecules. In some cases, the cleanup process may allow for sizeselection of nucleic acid molecules. In some cases, the cleanup processcomprises removing DNA-templated ligation products. In other cases, thecleanup process comprises RNAse to cleave the RNA strand, e.g., in aDNA-RNA duplex. In some cases, the cleanup process comprises a pulldownassay (e.g., biotin pulldown of a ligation handle). In some cases, thecleanup process comprises post-ligation exonuclease treatment. In somecases, the cleanup process comprises, blocking free 3′ ends on nucleicacid molecules, which may render them non-extendable by polymerase. Insome cases, the probe-linked nucleic acid molecules may be subjected tonucleic acid reactions, such as amplification (e.g., PCR). The amplifiedproducts will comprise sequences that are the same or substantially thesame as the barcode sequence and sequences of the target regions of thenucleic acid molecule from which they derive. The amplified products canbe traced back to particular nucleic acid molecules and, in some cases,to particular cells. This barcoding method may therefore facilitaterapid analysis of nucleic acid molecules through, for example,sequencing without the need for reverse transcription.

In some embodiments, a target-specific probe (e.g., the probe-linkedmolecules described herein) hybridized to a sample nucleic acid molecule(e.g., a cellular mRNA molecule) may be barcoded through combinatorialassembly of barcode segments using, e.g., a split-pool approach. Forexample, a plurality of permeabilized cells (or permeabilized nuclei orcell beads) are contacted with one or more target-specific probes asdescribed herein. Panel 11A of FIG. 11 depicts a target specific probehybridized to a target mRNA molecule 1100. The target-specific probe(s)may be configured using any suitable methodology described elsewhereherein (see, e.g., FIGS. 9-12, 14, 16, 17 , molecular inversion probes,etc.). For example, in some instances, a first probe comprising bindingsequence 1105 and adapter sequence 1106 and a second probe comprisingbinding sequence 1104 and adapter sequence 1103 is hybridized to targetnucleic acid molecule 1100 (e.g., a mRNA molecule) and the two probesare linked using, e.g., the enzymatic and/or chemical ligation schemesdescribed elsewhere herein to generate a probe linked nucleic acidmolecule 1120. Binding sequence 1105 is configured to hybridize totarget region 1101 of target nucleic acid molecule 1100 while bindingsequence 1104 is configured to hybridize to target region 1102 of targetnucleic acid molecule 1100. Adapter sequences 1106 and 1103 may eachoptionally comprise one or more functional sequences (e.g., a primersequence/primer binding sequence, a sequencing primer sequence (e.g., R1or R2), a partial sequencing primer sequence (e.g., partial R1 orpartial R2), a sequence configured to attach to the flow cell of asequencer (e.g., P5 or P7, or partial sequences thereof), a barcodesequence, UMI sequence, or complements of these sequences). Probe-linkednucleic acid molecule 1120 may then be barcoded using a combinatorialassembly of barcode sequence segments (i.e., barcode subunits). Forexample, in some embodiments, probe-linked nucleic acid molecule 1120 iscombinatorially barcoded using a split pool approach. In someembodiments, probe-linked nucleic acid molecule 1120 is combinatoriallybarcoded by successive addition of barcode sequence segments. Acombinatorial barcode sequence may be synthesized by various methodsincluding, for example, ligation, hybridization, nucleotidepolymerization, or a combination thereof.

Panel 11B shows addition of a first barcode sequence segment toprobe-linked nucleic acid molecule 1120. A partially double-strandednucleic acid barcode molecule comprising (i) a first strand 1108comprising a first barcode sequence segment and an adapter sequence and(ii) a second strand 1107 comprising a binding sequence is hybridized toprobe-linked nucleic acid molecule 1120. The binding sequence iscomplementary to at least a portion of adapter sequence 1106 such thatthe nucleic acid barcode molecule hybridizes to probe-linked nucleicacid molecule 1120. Strand 1108 is then attached to probe-linked nucleicacid molecule 1120 (e.g., using ligation and/or nucleic acid extension)to add the first barcode sequence segment.

Panel 11C shows addition of a second barcode sequence segment toprobe-linked nucleic acid molecule 1120 comprising the nucleic acidbarcode molecule comprising 1108. A partially double-stranded nucleicacid barcode molecule comprising (i) a first strand 1110 comprising afirst barcode sequence segment and an adapter sequence and (ii) a secondstrand 1109 comprising a binding sequence is hybridized to probe-linkednucleic acid molecule 1120 comprising the nucleic acid barcode moleculecomprising 1108. The binding sequence is complementary to at least aportion of the adapter sequence such that the nucleic acid barcodemolecule hybridizes to probe-linked nucleic acid molecule 1120comprising the nucleic acid barcode molecule comprising 1108. Strand1110 is then attached to probe-linked nucleic acid molecule 1120comprising the nucleic acid barcode molecule comprising 1108 (e.g.,using ligation and/or nucleic acid extension) to add the second barcodesequence segment.

Panel 11D shows addition of a third barcode sequence segment toprobe-linked nucleic acid molecule 1120 comprising 1108 and 1110. Apartially double-stranded nucleic acid barcode molecule comprising (i) afirst strand 1112 comprising a first barcode sequence segment and anadapter sequence and (ii) a second strand 1111 comprising a bindingsequence is hybridized to probe-linked nucleic acid molecule 1120comprising 1108 and 1110. The binding sequence is complementary to atleast a portion of the adapter sequence such that the nucleic acidbarcode molecule hybridizes to probe-linked nucleic acid molecule 1120comprising 1108 and 1110. Strand 1112 is then attached to probe-linkednucleic acid molecule 1120 comprising 1108 and 1110 (e.g., usingligation and/or nucleic acid extension) to add the third barcodesequence segment.

The combinatorial barcoding scheme described above can be implementedusing, e.g., a split-pool approach. For example, a plurality ofpermeabilized cells (or permeabilized nuclei or cell beads) comprising,e.g., probe-linked nucleic acid molecule 1120 (or any other probedescribed herein) may be partitioned into a first plurality ofpartitions (e.g., a plurality of wells) wherein each partition of theplurality of partitions comprises a different (i.e., unique) firstbarcode sequence segment. After addition of the first barcode sequencesegment, cells (or nucleic or cell beads) can be collected from thefirst plurality of partitioned and pooled and partitioned into a secondplurality of partitions (e.g., a plurality of wells) wherein eachpartition of the plurality of partitions comprises a different (i.e.,unique) second barcode sequence segment. Repeating this split-poolprocess allows the generation of barcodes comprising any suitable amountof barcode sequence segments. Combinatorial barcoding as describedherein may comprise at least 1, 2, 3, 4, 5, 6, 7, 8 or more operations(e.g., split-pool cycles). Combinatorial barcoding comprising multipleoperations may be useful, for example, in generation of greater barcodediversity and to synthesize a unique barcode sequence on nucleic acidmolecules derived from each single cell of a plurality of cells. Forexample, combinatorial barcoding comprising three operations, eachcomprising attachment of a unique nucleic acid sequence in each of 96partitions, will yield up to 884,736 unique barcode combinations. Cellsmay be partitioned such that at least one cell (or nuclei or cell bead)is present in each partition of a plurality of partitions. Cells may bepartitioned such that at least 1; 2; 3; 4; 5; 10; 20; 50; 100; 500;1,000; 5,000; 10,000; 100,000; 1,000,000; or more cells are present in asingle partition. Cells may be partitioned such that at most 1,000,000;100,000; 10,000; 5,000; 1,000; 500; 100; 50; 20; 10; 5; 4; 3; 2; or 1cell is present in a single partition. Cells may be partitioned in arandom configuration.

In some instances, the methods described herein are performed in a cellbead. See, e.g., U.S. Pat. Pub. 2018/0216162 and U.S. Pat. Pub.2019/0100632 for exemplary cell bead generation and processing methods.For example, in some embodiments, a cell bead comprising a cell isgenerated as described elsewhere herein. In some instances, the cellbead comprises, attached thereto (e.g., covalently attached to the cellbead polymer or cross-linked matrix), a plurality of nucleic acidmolecules comprising a poly-T sequence. In some instances, the nucleicacid molecules comprising a poly-T sequence are releasably attached tothe c el bead (e.g., via a labile bond as described elsewhere herein).Nucleic acid molecules comprising a poly-T sequence may also compriseone or more functional sequences (e.g., a primer sequence/primer bindingsequence, a sequencing primer sequence (e.g., R1 or R2), a partialsequencing primer sequence (e.g., partial R1 or partial R2), a sequenceconfigured to attach to the flow cell of a sequencer (e.g., P5 or P7, orpartial sequences thereof), a barcode sequence, UMI sequence, orcomplements of these sequences). Cells in the cell bead may then belysed to release cellular constituents, including mRNA moleculescomprising a poly-A tail. Alternatively, cells may be lysed prior to orconcurrent with cell bead generation (e.g., in droplets prior to orconcurrent with cell bead generation). Poly-A containing mRNA may thenbe hybridized to the poly-T sequence, thereby immobilizing mRNA in thecell bead. In some instances, captured mRNA is subjected to a reversetranscription reaction to convert captured mRNA into cDNA. In someinstances, the cDNA is single stranded. In other instances, the cDNA isdouble stranded. Nucleic acid molecules immobilized in cell beads, canthen be contacted by the probe molecules described herein and processedto detect cellular nucleic acid molecules (such as mRNA) as describedherein. In some instances, the cell bead is used to contain cellular DNAduring DNA denaturation by heat or chemical denaturation. The probesdescribed above can be used to target and detect DNA sequences,analogous to the description above.

Also provided herein are methods that may involve cell multiplexing.Cells may be processed, partitioned, and labeled. Processed cells may bepooled and nucleic acid molecules from the cells may be furtherprocessed. One or more of the processes may involve a nucleic acidreaction, barcoding, partitioning, and/or any combinations orderivatives thereof. One or more of the methods disclosed herein mayallow for cell multiplexing without the use of staining reagents and mayresult in improved occupancy of partitions. One or more of the processesmay involve hybridizing a probe to a target region of a nucleic acidmolecule of interest, barcoding the resultant complex, and performing anextension, denaturation, and amplification processes to provide nucleicacid molecules comprising a sequence the same or substantially the sameas or complementary to that of the target region of the nucleic acidmolecule of interest.

A multiplexing method may comprise hybridizing a first probe and asecond probe to first and second target regions of the nucleic acidmolecule, linking the first and second probes to provide a probe-linkednucleic acid molecule, and barcoding the probe-linked nucleic acidmolecule. One or more processes of the methods provided herein may beperformed within a partition such as a droplet or well.

In other cases, a multiplexing method may comprise hybridizing a firstprobe to a first target region of a nucleic acid molecule, barcoding thefirst probe within a first partition with a first barcode sequence,recovering the barcoded first probe from the partition, partitioning thefirst probe hybridized to the first target region of the nucleic acidmolecule within a second partition, hybridizing a second probe to asecond target region of the nucleic acid molecule within the secondpartition, and barcoding the first or second probe hybridized to thenucleic acid molecule with a second barcode sequence. In some cases, thefirst probe may comprise the first barcode sequence and barcoding with afirst barcode sequence within the first partition may be simultaneouswith hybridizing the first probe to the first target region. In somecases, the second probe may comprise the second barcode sequence andbarcoding with a second barcode sequence may be simultaneous withhybridizing the second probe to the second target region. In some cases,the first probe may be linked to the second probe (e.g., via a chemicalor enzymatic ligation process, as described herein). The first andsecond probes may be linked to one another within the second partitionor outside of the second partition. This process may be repeated for aplurality of nucleic acid molecules (e.g., nucleic acid moleculesincluded within cells, such as fixed cells or cell beads) across aplurality of first partitions and a plurality of second partitions. Eachfirst partition of the plurality of first partitions may comprise adifferent first barcode sequence of a plurality of first barcodesequences, and each second partition of the plurality of secondpartitions may comprise a different second barcode sequence of aplurality of second barcode sequences. First barcode sequences may becomponents of first nucleic acid barcode molecules coupled to a firstplurality of beads, while second barcode sequences may be components ofsecond nucleic acid barcode molecules coupled to a second plurality ofbeads (e.g., as described herein). The plurality of first partitions maybe wells, while the second plurality of partitions may be droplets(e.g., as described herein).

In an aspect, a multiplexing method provided herein comprises, (i)fixing a plurality of cells or cell beads, (ii) performing a firstpartitioning of the plurality of cells or cell beads, (iii) barcoding aplurality of nucleic acid molecules within the plurality of cells orcell beads to provide a plurality of labeled cells or cell beadscomprising barcoded nucleic acid molecules, (iv) pooling the pluralityof labeled cells or cell beads comprising the barcoded nucleic acidmolecules, (v) performing a second partitioning of said plurality oflabeled cells or cell beads comprising the barcoded nucleic acidmolecules, and (vi) performing a second barcoding of the barcodednucleic acid molecules to produce multiplexed barcoded nucleic acidmolecules.

In some embodiments, the cell or cell bead may be processed to barcodethe cell. The cell bead may comprise a cell. In some embodiments, thecell may be alive. In some embodiments, the cell may be fixed using afixative agent such as paraformaldehyde, formaldehyde, ethanol,methanol, etc. In some cases, the fixed cell may also be permeabilized.In some embodiments, a plurality of cells (e.g., fixed, permeabilizedcells) may be partitioned among a plurality of partitions. In somecases, a cell (e.g., a fixed cell) is permeabilized within a partition.Within the plurality of partitions, the plurality of cells (e.g., fixed,permeabilized cells) may be barcoded. In some embodiments, nucleic acidmolecules within the plurality of cells (e.g., fixed, permeabilizedcells) may be barcoded.

In some cases, the method may comprise providing a sample comprising anucleic acid molecule (e.g., an RNA molecule) having adjacent first andsecond target regions; a first probe having a first probe sequence thatis complementary to the first target region and a second probe sequence;and a second probe having a third probe sequence that is complementaryto the second target region. The first and third probe sequences mayalso comprise first and second reactive moieties, respectively. Uponhybridization of the first probe sequence of the first probe to thefirst target region of the nucleic acid molecule, and hybridization ofthe third probe sequence of the second probe to the second target regionof the nucleic acid molecule, the reactive moieties may be adjacent toone another. Subsequent reaction between the adjacent reactive moietiesunder sufficient conditions may link the first and second probes toyield a probe-linked nucleic acid molecule. The probe-linked nucleicacid molecule may also be referred to as a probe-ligated nucleic acidmolecule. The probe-linked nucleic acid molecule may then be barcodedwith a barcode sequence of a nucleic acid barcode molecule to provide abarcoded probe-linked nucleic acid molecule. Barcoding may be achievedby hybridizing a binding sequence of the nucleic acid barcode moleculeto the second probe sequence of the first probe of the probe-linkednucleic acid molecule. In some cases, the first probe or the secondprobe may comprise a barcode sequence. In some cases, both the firstprobe and the second probe comprise a barcode sequence. In some cases,the first probe and the second probe may be parts of the same probe andmay be connected. In some cases, the first probe and the second probemay be parts of a linear probe that forms a circularized nucleic acidproduct upon hybridization of the first probe and the second probe withthe target nucleic acid molecule. The barcoded nucleic acid molecule maybe subjected to amplification reactions to yield an amplified productcomprising the first and second target regions and the barcode sequenceor sequences complementary to these sequences. One or more processes maybe performed within a partition such as a droplet or well.

In some cases, the method may comprise providing a sample comprising anucleic acid molecule (e.g., an RNA molecule) having first and secondtarget regions; a first probe having a first probe sequence that iscomplementary to the first target region and a second probe sequence;and a second probe having a third probe sequence that is complementaryto the second target region. The first and second target regions may beadjacent to one another. Alternatively, the first and second targetregions may be separated by a gap region of at least one nucleotide,such as at least 1, 10, 50, or 100 nucleotides. The first probe sequenceof the first probe may hybridize to the first target region of thenucleic acid molecule, and the third probe sequence of the second probemay hybridize to the second target region of the nucleic acid moleculeto provide a probe-associated nucleic acid molecule. Subsequent tohybridization of the first probe sequence of the first probe to thefirst target region of the nucleic acid molecule, and hybridization ofthe third probe sequence of the second probe to the second target regionof the nucleic acid molecule, the first and second probes may be linkedto one another (e.g., via a chemical or enzymatic ligation process, asdescribed herein). For example, the first probe may comprise a firstreactive moiety and the second probe may comprise a second reactivemoiety, and the first and second reactive moieties may react undersufficient conditions may link the first and second probes to yield aprobe-linked nucleic acid molecule. The probe-linked nucleic acidmolecule may also be referred to as a probe-ligated nucleic acidmolecule. The probe-linked nucleic acid molecule may then be barcodedwith a barcode sequence of a nucleic acid barcode molecule to provide abarcoded probe-linked nucleic acid molecule. Alternatively, theprobe-associated nucleic acid molecule may be barcoded to provide abarcoded probe-associated nucleic acid molecule. Barcoding may beachieved by hybridizing a binding sequence of the nucleic acid barcodemolecule to the second probe sequence of the first probe of theprobe-linked nucleic acid molecule. In some cases, the first probe orthe second probe may comprise a barcode sequence. In some cases, boththe first probe and the second probe comprise a barcode sequence. Insome cases, the first probe and the second probe may be parts of thesame probe and may be connected (e.g., by one or more linking sequences,as described herein). In some cases, the first probe and the secondprobe may be parts of a linear probe that forms a circularized nucleicacid product upon hybridization of the first probe and the second probewith the target nucleic acid molecule. The barcoded nucleic acidmolecule may be subjected to amplification reactions to yield anamplified product comprising the first and second target regions and thebarcode sequence or sequences complementary to these sequences. One ormore processes may be performed within a partition such as a droplet orwell.

In some cases, a second barcoding operation may be performed to generatemultiplexed barcoded nucleic acid molecules. The operation may comprise(i) pooling a plurality of cells, wherein a cell of the plurality ofcells comprises a barcoded nucleic acid molecule, (ii) partitioning theplurality of cells, and (iii) barcoding the barcoded nucleic acidmolecule to produce a multiplexed barcoded nucleic acid molecule. One ormore processes may be performed within a partition such as a droplet orwell. In some cases, pooling of the cells comprising the barcodednucleic acid molecule may be performed in a container, such as a vesselor a tube. The pooled cells may then be further partitioned. Thepartition may comprise conditions sufficient to barcode the barcodednucleic acid molecule to generate a multiplexed barcoded nucleic acidmolecule. In some cases, the conditions comprise a barcode molecule andan enzyme. In some cases, the conditions comprise a barcode molecule, anadapter molecule, and an enzyme. The enzyme may be a ligase, polymerase,or any other suitable enzyme or combinations of enzymes. In onenon-limiting example, a cell comprising a barcoded nucleic acid moleculemay be partitioned with an adapter molecule comprising a probe-bindingsequence and a barcode-binding sequence. In some cases, the partitionalso comprises a barcode molecule and an enzyme. In some cases, theprobe-binding sequence of the adapter molecule may hybridize with asequence on the barcoded nucleic acid molecule. In some cases, thebarcode-binding sequence of the adapter molecule may hybridize with asequence of the barcode molecule. The barcode molecule may then beadjacent to the barcoded nucleic acid molecule. The barcode molecule maythen be ligated (e.g., using an enzyme) to the barcoded nucleic acidmolecule, generating a multiplexed barcoded nucleic acid molecule. Themultiplexed barcoded nucleic acid molecules may be used to determinecellular occupancy in a partition and may provide a method for improvedcellular loading, increased occupancy, determination ofmultiply-occupied partitions, and may obviate the need for cell stainingreagents.

In some embodiments, the probes described herein (e.g., 1206, 2014,1305, 1310, 1340, 1706, etc.) comprise a barcode sequence. In someinstances, target nucleic acid molecules (e.g., mRNA molecules) within acell (e.g., a fixed and/or permeabilized cell) are contacted withbarcoded probes to facilitate cell multiplexing and/or more robust andefficient analysis of cellular polynucleotides. For example, FIGS. 22A-Cschematically illustrates a method for improved processing nucleic acidmolecules from a cell. Panel 22A illustrates exemplary barcoded probes(2201, 2202) that may be utilized with the methods described herein.Probe 2201 comprises probe sequences 2210, barcode sequence 2211, andadapter sequence 2212. Probe 2202 comprises probe sequences 2220,barcode sequence 2221, and adapter sequence 2222. Probe sequences 2210and 2220 are complementary to a target region of a cellularpolynucleotide (e.g., mRNA molecule) as described elsewhere herein. Insome instances, probes 2201 and/or 2202 may comprise a reactive moiety(e.g., click chemistry moiety) as described elsewhere herein. In somecases, probes 2201 and 2202 are ligated chemically (e.g., clickchemistry), and in other cases, enzymatically (e.g., a ligase, such asSplintR or T4 ligase) to generate a probe-linked nucleic acid moleculecomprising sequences 2212, 2211, 2210, 2220, 2221, and 2222.

Panel A of FIG. 22B illustrates schematically an exemplary partitioningand processing scheme. A plurality of cells 2203 may be fixed and/orpermeabilized in process 2230 to provide processed cells 2204. In someinstances, cells 2203 are first partitioned followed by in-partitionfixation and/or permeabilization. In process 2240, cells 2203 or 2204are partitioned into a plurality of partitions, e.g., into wells of amultiwell array 2205. In some instances, each partition (e.g., well of amultiwell array) comprises a single cell. In other embodiments, eachpartition (e.g., well of a multiwell array) comprises a plurality ofcells. For example, cells may be partitioned such that are 1; 2; 3; 4;5; 10; 20; 50; 100; 500; 1,000; 5,000; 10,000; 100,000; 1,000,000; ormore cells are present in a single partition. Cells may be partitionedsuch that at least 1; 2; 3; 4; 5; 10; 20; 50; 100; 500; 1,000; 5,000;10,000; 100,000; 1,000,000; or more cells are present in a singlepartition. Cells may be partitioned such that at most 1,000,000;100,000; 10,000; 5,000; 1,000; 500; 100; 50; 20; 10; 5; 4; 3; 2; or 1cell is present in a single partition.

Barcoded probes are distributed into the partitions (either prior to,concurrent with, or subsequent to cell partitioning) such that eachpartition comprises probes comprising a partition-specific probebarcode. For example, in some instances, barcoded probes (e.g., 2201 and2202) are partitioned into rows and columns as described in FIG. 22B,Panel B. In FIG. 22B, Panel B, barcoded probes (e.g., 2201) aredistributed such that each well in a column of microwell array 2205comprises a common barcode sequence (e.g., 2211) while each well indifferent columns of microwell array 2205 comprises probes (e.g., 2201)comprising different barcode sequences (e.g., 2211). For example, eachwell in column 1 will comprise probe molecule 2201 a comprising targetsequence 2210 and column barcode sequence 2211 a. Likewise, each well incolumn 2 will comprise probe molecule 2201 b comprising target sequence2210 and column barcode sequence 2211 b; while each well in column 3will comprise probe molecule 2201 c comprising target sequence 2210 andcolumn barcode sequence 2211 c, etc. Similarly, probe 2202 isdistributed such that each well in a row of microwell array 2205comprises a common barcode sequence 2221 while each well in differentrows of microwell array 2205 will comprise probes 2202 comprisingdifferent barcode sequences 2221. For example, each well in row 1 willcomprise probe molecule 2202 a comprising target sequence 2220 and rowbarcode sequence 2221 a. Likewise, each well in row 2 will compriseprobe molecule 2202 b comprising target sequence 2220 and row barcodesequence 2221 b while each well in row 3 will comprise probe molecule2202 c comprising target sequence 2220 and row barcode sequence 2221 c,etc. Thus, each well of microwell array 2205 comprises a uniquepartition barcode comprising column barcode sequence (e.g., 2211 a-f)and a row barcode sequence (e.g., 2221 a-c).

In this fashion, barcoded probe molecules specific for a panel of targetpolynucleotides (e.g., a panel of mRNA molecules) can be co-partitionedwith cells, e.g., in the column and row format described above. Forexample, each well in column 1 may comprise a plurality of barcodedprobe molecules (e.g., 2201) comprising a plurality of target sequences(e.g., 2210 a, 2210 b, 2210 c, etc.) complementary to a plurality ofcellular polynucleotides (e.g., a panel of mRNA molecules) and columnbarcode sequence 2211 a. Likewise, each well in column 2 will comprise aplurality of probe molecules (e.g., 2201) comprising a plurality oftarget sequences (e.g., 2210 a, 2210 b, 2210 c, etc.) complementary tothe plurality of cellular polynucleotides (e.g., panel of mRNAmolecules), but with column barcode sequence 2211 b, etc. Similarly,each well in row 1 will comprise a plurality of barcoded probe molecules(e.g., 2202) comprising a plurality of target sequences (e.g., 2220 a,2220 b, 2220 c, etc.) complementary to the plurality of cellularpolynucleotides (e.g., the panel of mRNA molecules) and row barcodesequence 2221 a. Likewise, each row in column 2 will comprise aplurality of barcoded probe molecules (e.g., 2202) comprising aplurality of target sequences (e.g., 2220 a, 2220 b, 2220 c, etc.)complementary to the plurality of cellular polynucleotides (e.g., thepanel of mRNA molecules) and row barcode sequence 2221 b, etc.

In some instances, only one of probe 2201 or 2202 will comprise abarcode sequence and probes 2201 and 2202 are partitioned such that eachpartition comprises a unique barcode sequence.

After co-partitioning of cells (e.g., 2204) and barcoded probes (e.g.,2201 and 2202), probes are hybridized to their target nucleic acid,unbound probes are optionally washed away, and probes are enzymatically(e.g., by ligation) or chemically (e.g., click chemistry) joined aspreviously described (see, e.g., FIG. 12 and accompanying text). In someinstances, probes 2201 and 2202 are subjected to a gap-fill reaction aspreviously described (see, e.g., FIG. 16 and accompanying text). Asschematically shown in FIG. 22C, Panel A, after processing of barcodedprobes (e.g., chemical or enzymatic ligation), cells are pooled inprocess 2250 to provide a pooled plurality of cells 2206. The pooledplurality of cells 2206 may then be partitioned in process 2260 into asecond set of partitions 2207 (e.g., droplets or wells) such that atleast some partitions comprise (1) one or more cells of the pooledplurality of cells; and (2) nucleic acid barcode molecules. The cellsare then processed to barcode the linked probe molecules as describedelsewhere herein. In some instances, each partition may comprise aunique nucleic acid barcode molecule. Partitions 2207 may also compriselysis reagents for lysis and release of the barcoded probes from cells.In one example, as shown in FIG. 22C, Panel B, a partition 2208 of theplurality of partitions 2207 comprises a bead comprising nucleic acidbarcode molecules (e.g., 2270) attached thereto. In some instances, thebead is a gel bead as described elsewhere herein.

As shown in FIG. 22C, Panel B, nucleic acid barcode molecule 2270comprises an adapter sequence 2271, and a barcode sequence 2272 (whichoptionally may comprise a UMI sequence), and binding sequence 2271,which is complementary to adapter sequence 2212 of the linked probe2290. The adapter sequence 2271 may comprise one or more functionalsequences (e.g., a primer sequence/primer binding sequence, a sequencingprimer sequence (e.g., R1 or R2), a partial sequencing primer sequence(e.g., partial R1 or partial R2), a sequence configured to attach to theflow cell of a sequencer (e.g., P5 or P7, or partial sequences thereof),or complements of these sequences). Nucleic acid barcode molecule 2270is then hybridized to sequence 2212 of the probe-linked nucleic acidmolecule 2290. A barcoded probe-linked nucleic acid molecule is thengenerated using, e.g., a nucleic acid extension reaction and/or ligationreaction as described previously. The barcoded probe-linked nucleic acidmolecule will comprise both the probe-specific barcode (e.g., 2211and/or 2221) as well as the partition specific barcode 2272. Because ofthe presence of both the probe-specific barcode(s) (e.g., 2211 and/or2221) and the partition specific barcode 2272, partitions comprisingcell multiplets (e.g., cell doublets, triplets, etc.) could then becomputationally deconvolved into single cells and this data retainedwhere it typically would be discarded. Thus, in some instances, cellsare “overloaded” into partitions using conditions such that a higherprobability of cell multiplets (2,3,4,5+ cells per partition) areformed, wherein target libraries of these cell multiplets arecomputationally deconvolved into single cells.

After the partition-based barcoding step (FIG. 22C, Panel B), thecontents of the partitions 2207 may be pooled and the barcodedprobe-linked nucleic acid molecules may be duplicated or amplified by,for example, one or more amplification reactions, which may in someinstances be isothermal. The amplification reactions may comprisepolymerase chain reactions (PCR) and may involve the use of one or moreprimers or polymerases. The one or more primers may comprise one or morefunctional sequences (e.g., a primer sequence/primer binding sequence, asequencing primer sequence (e.g., R1 or R2), a partial sequencing primersequence (e.g., partial R1 or partial R2), a sequence configured toattach to the flow cell of a sequencer (e.g., P5 or P7, or partialsequences thereof), etc.) and may facilitate addition of said one ormore functional sequences to the extended nucleic acid molecule. Thebarcoded probe-linked nucleic acid molecule, or a derivative thereof,may be detected via nucleic acid sequencing (e.g., as described herein).

Systems and Methods for Sample Compartmentalization

In an aspect, the systems and methods described herein provide for thecompartmentalization, depositing, or partitioning of one or moreparticles (e.g., biological particles, macromolecular constituents ofbiological particles, beads, reagents, etc.) into discrete compartmentsor partitions (referred to interchangeably herein as partitions), whereeach partition maintains separation of its own contents from thecontents of other partitions. The partition can be a droplet in anemulsion. A partition may comprise one or more other partitions.

A partition may include one or more particles. A partition may includeone or more types of particles. For example, a partition of the presentdisclosure may comprise one or more biological particles and/ormacromolecular constituents thereof. A partition may comprise one ormore gel beads. A partition may comprise one or more cell beads. Apartition may include a single gel bead, a single cell bead, or both asingle cell bead and single gel bead. A partition may include one ormore reagents. Alternatively, a partition may be unoccupied. Forexample, a partition may not comprise a bead. A cell bead can be abiological particle and/or one or more of its macromolecularconstituents encased inside of a gel or polymer matrix, such as viapolymerization of a droplet containing the biological particle andprecursors capable of being polymerized or gelled. Unique identifiers,such as barcodes, may be injected into the droplets previous to,subsequent to, or concurrently with droplet generation, such as via amicrocapsule (e.g., bead), as described elsewhere herein. Microfluidicchannel networks (e.g., on a chip) can be utilized to generatepartitions as described herein. Alternative mechanisms may also beemployed in the partitioning of individual biological particles,including porous membranes through which aqueous mixtures of cells areextruded into non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions maycomprise, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionsmay comprise a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions may beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. PatentApplication Publication No. 2014/0155295, which is entirely incorporatedherein by reference for all purposes. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are describedin, for example, U.S. Patent Application Publication No. 2010/0105112,which is entirely incorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual particlesto discrete partitions may in one non-limiting example be accomplishedby introducing a flowing stream of particles in an aqueous fluid into aflowing stream of a non-aqueous fluid, such that droplets are generatedat the junction of the two streams. Fluid properties (e.g., fluid flowrates, fluid viscosities, etc.), particle properties (e.g., volumefraction, particle size, particle concentration, etc.), microfluidicarchitectures (e.g., channel geometry, etc.), and other parameters maybe adjusted to control the occupancy of the resulting partitions (e.g.,number of biological particles per partition, number of beads perpartition, etc.). For example, partition occupancy can be controlled byproviding the aqueous stream at a certain concentration and/or flow rateof particles. To generate single biological particle partitions, therelative flow rates of the immiscible fluids can be selected such that,on average, the partitions may contain less than one biological particleper partition in order to ensure that those partitions that are occupiedare primarily singly occupied. In some cases, partitions among aplurality of partitions may contain at most one biological particle(e.g., bead, DNA, cell or cellular material). In some embodiments, thevarious parameters (e.g., fluid properties, particle properties,microfluidic architectures, etc.) may be selected or adjusted such thata majority of partitions are occupied, for example, allowing for only asmall percentage of unoccupied partitions. The flows and channelarchitectures can be controlled as to ensure a given number of singlyoccupied partitions, less than a certain level of unoccupied partitionsand/or less than a certain level of multiply occupied partitions.

FIG. 1 shows an example of a microfluidic channel structure 100 forpartitioning individual biological particles. The channel structure 100can include channel segments 102, 104, 106 and 108 communicating at achannel junction 110. In operation, a first aqueous fluid 112 thatincludes suspended biological particles (or cells) 114 may betransported along channel segment 102 into junction 110, while a secondfluid 116 that is immiscible with the aqueous fluid 112 is delivered tothe junction 110 from each of channel segments 104 and 106 to creatediscrete droplets 118, 120 of the first aqueous fluid 112 flowing intochannel segment 108, and flowing away from junction 110. The channelsegment 108 may be fluidically coupled to an outlet reservoir where thediscrete droplets can be stored and/or harvested. A discrete dropletgenerated may include an individual biological particle 114 (such asdroplets 118). A discrete droplet generated may include more than oneindividual biological particle 114 (not shown in FIG. 1 ). A discretedroplet may contain no biological particle 114 (such as droplet 120).Each discrete partition may maintain separation of its own contents(e.g., individual biological particle 114) from the contents of otherpartitions.

The second fluid 116 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets118, 120. Examples of particularly useful partitioning fluids andfluorosurfactants are described, for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 100 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junction.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying particles (e.g., biological particles,cell beads, and/or gel beads) that meet at a channel junction. Fluid maybe directed to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 118, containing one or more biological particles 114,and (2) unoccupied droplets 120, not containing any biological particles114. Occupied droplets 118 may comprise singly occupied droplets (havingone biological particle) and multiply occupied droplets (having morethan one biological particle). As described elsewhere herein, in somecases, the majority of occupied partitions can include no more than onebiological particle per occupied partition and some of the generatedpartitions can be unoccupied (of any biological particle). In somecases, though, some of the occupied partitions may include more than onebiological particle. In some cases, the partitioning process may becontrolled such that fewer than about 25% of the occupied partitionscontain more than one biological particle, and in many cases, fewer thanabout 20% of the occupied partitions have more than one biologicalparticle, while in some cases, fewer than about 10% or even fewer thanabout 5% of the occupied partitions include more than one biologicalparticle per partition.

In some cases, it may be desirable to minimize the creation of excessivenumbers of empty partitions, such as to reduce costs and/or increaseefficiency. While this minimization may be achieved by providing asufficient number of biological particles (e.g., biological particles114) at the partitioning junction 110, such as to ensure that at leastone biological particle is encapsulated in a partition, the Poissoniandistribution may expectedly increase the number of partitions thatinclude multiple biological particles. As such, where singly occupiedpartitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% orless of the generated partitions can be unoccupied.

In some cases, the flow of one or more of the biological particles(e.g., in channel segment 102), or other fluids directed into thepartitioning junction (e.g., in channel segments 104, 106) can becontrolled such that, in many cases, no more than about 50% of thegenerated partitions, no more than about 25% of the generatedpartitions, or no more than about 10% of the generated partitions areunoccupied. These flows can be controlled so as to present anon-Poissonian distribution of single-occupied partitions whileproviding lower levels of unoccupied partitions. The above noted rangesof unoccupied partitions can be achieved while still providing any ofthe single occupancy rates described above. For example, in many cases,the use of the systems and methods described herein can create resultingpartitions that have multiple occupancy rates of less than about 25%,less than about 20%, less than about 15%, less than about 10%, and inmany cases, less than about 5%, while having unoccupied partitions ofless than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 10%, less than about 5%, or less.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both biological particles andadditional reagents, including, but not limited to, microcapsules orbeads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g.,oligonucleotides) (described in relation to FIG. 2 ). The occupiedpartitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 99% of the occupied partitions) can include both amicrocapsule (e.g., bead) comprising barcoded nucleic acid molecules anda biological particle.

In another aspect, in addition to or as an alternative to droplet basedpartitioning, biological particles may be encapsulated within amicrocapsule that comprises an outer shell, layer or porous matrix inwhich is entrained one or more individual biological particles or smallgroups of biological particles. The microcapsule may include otherreagents. Encapsulation of biological particles may be performed by avariety of processes. Such processes may combine an aqueous fluidcontaining the biological particles with a polymeric precursor materialthat may be capable of being formed into a gel or other solid orsemi-solid matrix upon application of a particular stimulus to thepolymer precursor. Such stimuli can include, for example, thermalstimuli (e.g., either heating or cooling), photo-stimuli (e.g., throughphoto-curing), chemical stimuli (e.g., through crosslinking,polymerization initiation of the precursor (e.g., through addedinitiators)), mechanical stimuli, or a combination thereof.

Preparation of microcapsules comprising biological particles may beperformed by a variety of methods. For example, air knife droplet oraerosol generators may be used to dispense droplets of precursor fluidsinto gelling solutions in order to form microcapsules that includeindividual biological particles or small groups of biological particles.Likewise, membrane based encapsulation systems may be used to generatemicrocapsules comprising encapsulated biological particles as describedherein. Microfluidic systems of the present disclosure, such as thatshown in FIG. 1 , may be readily used in encapsulating cells asdescribed herein. In particular, and with reference to FIG. 1 , theaqueous fluid 112 comprising (i) the biological particles 114 and (ii)the polymer precursor material (not shown) is flowed into channeljunction 110, where it is partitioned into droplets 118, 120 through theflow of non-aqueous fluid 116. In the case of encapsulation methods,non-aqueous fluid 116 may also include an initiator (not shown) to causepolymerization and/or crosslinking of the polymer precursor to form themicrocapsule that includes the entrained biological particles. Examplesof polymer precursor/initiator pairs include those described in U.S.Patent Application Publication No. 2014/0378345, which is entirelyincorporated herein by reference for all purposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, such as a linear polyacrylamide, PEG, orother linear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams 116 in channel segments 104 and 106, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110, during formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous fluid 112 comprising thelinear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets 118, 120, resulting in the formationof gel (e.g., hydrogel) microcapsules, as solid or semi-solid beads orparticles entraining the cells 114. Although described in terms ofpolyacrylamide encapsulation, other ‘activatable’ encapsulationcompositions may also be employed in the context of the methods andcompositions described herein. For example, formation of alginatedroplets followed by exposure to divalent metal ions (e.g., Ca²⁺ ions),can be used as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling (e.g., upon cooling, etc.).

In some cases, encapsulated biological particles can be selectivelyreleasable from the microcapsule, such as through passage of time orupon application of a particular stimulus, that degrades themicrocapsule sufficiently to allow the biological particles (e.g.,cell), or its other contents to be released from the microcapsule, suchas into a partition (e.g., droplet). For example, in the case of thepolyacrylamide polymer described above, degradation of the microcapsulemay be accomplished through the introduction of an appropriate reducingagent, such as DTT or the like, to cleave disulfide bonds thatcross-link the polymer matrix. See, for example, U.S. Patent ApplicationPublication No. 2014/0378345, which is entirely incorporated herein byreference for all purposes.

The biological particle can be subjected to other conditions sufficientto polymerize or gel the precursors. The conditions sufficient topolymerize or gel the precursors may comprise exposure to heating,cooling, electromagnetic radiation, and/or light. The conditionssufficient to polymerize or gel the precursors may comprise anyconditions sufficient to polymerize or gel the precursors. Followingpolymerization or gelling, a polymer or gel may be formed around thebiological particle. The polymer or gel may be diffusively permeable tochemical or biochemical reagents. The polymer or gel may be diffusivelyimpermeable to macromolecular constituents of the biological particle.In this manner, the polymer or gel may act to allow the biologicalparticle to be subjected to chemical or biochemical operations whilespatially confining the macromolecular constituents to a region of thedroplet defined by the polymer or gel. The polymer or gel may includeone or more of disulfide cross-linked polyacrylamide, agarose, alginate,polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate,PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronicacid, collagen, fibrin, gelatin, or elastin. The polymer or gel maycomprise any other polymer or gel.

The polymer or gel may be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids or otheranalytes. The polymer or gel may be polymerized or gelled via a passivemechanism. The polymer or gel may be stable in alkaline conditions or atelevated temperature. The polymer or gel may have mechanical propertiessimilar to the mechanical properties of the bead. For instance, thepolymer or gel may be of a similar size to the bead. The polymer or gelmay have a mechanical strength (e.g. tensile strength) similar to thatof the bead. The polymer or gel may be of a lower density than an oil.The polymer or gel may be of a density that is roughly similar to thatof a buffer. The polymer or gel may have a tunable pore size. The poresize may be chosen to, for instance, retain denatured nucleic acids. Thepore size may be chosen to maintain diffusive permeability to exogenouschemicals such as sodium hydroxide (NaOH) and/or endogenous chemicalssuch as inhibitors. The polymer or gel may be biocompatible. The polymeror gel may maintain or enhance cell viability. The polymer or gel may bebiochemically compatible. The polymer or gel may be polymerized and/ordepolymerized thermally, chemically, enzymatically, and/or optically.

The polymer may comprise poly(acrylamide-co-acrylic acid) crosslinkedwith disulfide linkages. The preparation of the polymer may comprise atwo-step reaction. In the first activation step,poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent toconvert carboxylic acids to esters. For instance, thepoly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking step, the ester formed in the first step may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twosteps, the biological particle may be surrounded by polyacrylamidestrands linked together by disulfide bridges. In this manner, thebiological particle may be encased inside of or comprise a gel or matrix(e.g., polymer matrix) to form a “cell bead.” A cell bead can containbiological particles (e.g., a cell) or macromolecular constituents(e.g., RNA, DNA, proteins, etc.) of biological particles. A cell beadmay include a single cell or multiple cells, or a derivative of thesingle cell or multiple cells. For example after lysing and washing thecells, inhibitory components from cell lysates can be washed away andthe macromolecular constituents can be bound as cell beads. Systems andmethods disclosed herein can be applicable to both cell beads (and/ordroplets or other partitions) containing biological particles and cellbeads (and/or droplets or other partitions) containing macromolecularconstituents of biological particles.

Encapsulated biological particles can provide certain potentialadvantages of being more storable and more portable than droplet-basedpartitioned biological particles. Furthermore, in some cases, it may bedesirable to allow biological particles to incubate for a select periodof time before analysis, such as in order to characterize changes insuch biological particles over time, either in the presence or absenceof different stimuli. In such cases, encapsulation may allow for longerincubation than partitioning in emulsion droplets, although in somecases, droplet partitioned biological particles may also be incubatedfor different periods of time, e.g., at least 10 seconds, at least 30seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, atleast 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours,or at least 10 hours or more. The encapsulation of biological particlesmay constitute the partitioning of the biological particles into whichother reagents are co-partitioned. Alternatively or in addition,encapsulated biological particles may be readily deposited into otherpartitions (e.g., droplets) as described above.

Beads

A partition may comprise one or more unique identifiers, such asbarcodes. Barcodes may be previously, subsequently or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned biological particle. For example, barcodes may be injectedinto droplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual biological particle to the particular partition. Barcodes maybe delivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can comprise a bead.Beads are described in further detail below.

In some cases, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule and then released from themicrocapsule. Release of the barcoded nucleic acid molecules can bepassive (e.g., by diffusion out of the microcapsule). In addition oralternatively, release from the microcapsule can be upon application ofa stimulus which allows the barcoded nucleic acid nucleic acid moleculesto dissociate or to be released from the microcapsule. Such stimulus maydisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

FIG. 2 shows an example of a microfluidic channel structure 200 fordelivering barcode carrying beads to droplets. The channel structure 200can include channel segments 201, 202, 204, 206 and 208 communicating ata channel junction 210. In operation, the channel segment 201 maytransport an aqueous fluid 212 that includes a plurality of beads 214(e.g., with nucleic acid molecules, oligonucleotides, molecular tags)along the channel segment 201 into junction 210. The plurality of beads214 may be sourced from a suspension of beads. For example, the channelsegment 201 may be connected to a reservoir comprising an aqueoussuspension of beads 214. The channel segment 202 may transport theaqueous fluid 212 that includes a plurality of biological particles 216along the channel segment 202 into junction 210. The plurality ofbiological particles 216 may be sourced from a suspension of biologicalparticles. For example, the channel segment 202 may be connected to areservoir comprising an aqueous suspension of biological particles 216.In some instances, the aqueous fluid 212 in either the first channelsegment 201 or the second channel segment 202, or in both segments, caninclude one or more reagents, as further described below. A second fluid218 that is immiscible with the aqueous fluid 212 (e.g., oil) can bedelivered to the junction 210 from each of channel segments 204 and 206.Upon meeting of the aqueous fluid 212 from each of channel segments 201and 202 and the second fluid 218 from each of channel segments 204 and206 at the channel junction 210, the aqueous fluid 212 can bepartitioned as discrete droplets 220 in the second fluid 218 and flowaway from the junction 210 along channel segment 208. The channelsegment 208 may deliver the discrete droplets to an outlet reservoirfluidly coupled to the channel segment 208, where they may be harvested.

As an alternative, the channel segments 201 and 202 may meet at anotherjunction upstream of the junction 210. At such junction, beads andbiological particles may form a mixture that is directed along anotherchannel to the junction 210 to yield droplets 220. The mixture mayprovide the beads and biological particles in an alternating fashion,such that, for example, a droplet comprises a single bead and a singlebiological particle.

Beads, biological particles and droplets may flow along channels atsubstantially regular flow profiles (e.g., at regular flow rates). Suchregular flow profiles may permit a droplet to include a single bead anda single biological particle. Such regular flow profiles may permit thedroplets to have an occupancy (e.g., droplets having beads andbiological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95%. Such regular flow profiles and devices that maybe used to provide such regular flow profiles are provided in, forexample, U.S. Patent Publication No. 2015/0292988, which is entirelyincorporated herein by reference.

The second fluid 218 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets220.

A discrete droplet that is generated may include an individualbiological particle 216. A discrete droplet that is generated mayinclude a barcode or other reagent carrying bead 214. A discrete dropletgenerated may include both an individual biological particle and abarcode carrying bead, such as droplets 220. In some instances, adiscrete droplet may include more than one individual biologicalparticle or no biological particle. In some instances, a discretedroplet may include more than one bead or no bead. A discrete dropletmay be unoccupied (e.g., no beads, no biological particles).

Beneficially, a discrete droplet partitioning a biological particle anda barcode carrying bead may effectively allow the attribution of thebarcode to macromolecular constituents of the biological particle withinthe partition. The contents of a partition may remain discrete from thecontents of other partitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 200 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

A bead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead. A gel bead may be formed from molecularprecursors, such as a polymeric or monomeric species. A semi-solid beadmay be a liposomal bead. Solid beads may comprise metals including ironoxide, gold, and silver. In some cases, the bead may be a silica bead.In some cases, the bead can be rigid. In other cases, the bead may beflexible and/or compressible.

A bead may be of any suitable shape. Examples of bead shapes include,but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, and variations thereof.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be at least about 10 nanometers (nm), 100 nm, 500nm, 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm,70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or greater. In somecases, a bead may have a diameter of less than about 10 nm, 100 nm, 500nm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm,90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or less. In some cases, a bead mayhave a diameter in the range of about 40-75 μm, 30-75 μm, 20-75 μm,40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500μm.

In certain aspects, beads can be provided as a population or pluralityof beads having a relatively monodisperse size distribution. Where itmay be desirable to provide relatively consistent amounts of reagentswithin partitions, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, lessthan 5%, or less.

A bead may comprise natural and/or synthetic materials. For example, abead can comprise a natural polymer, a synthetic polymer or both naturaland synthetic polymers. Examples of natural polymers include proteinsand sugars such as deoxyribonucleic acid, rubber, cellulose, starch(e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silks,polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/orcombinations (e.g., co-polymers) thereof. Beads may also be formed frommaterials other than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some instances, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the molecular precursors. In some cases, a precursormay be an already polymerized species capable of undergoing furtherpolymerization via, for example, a chemical cross-linkage. In somecases, a precursor can comprise one or more of an acrylamide or amethacrylamide monomer, oligomer, or polymer. In some cases, the beadmay comprise prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads may be prepared usingprepolymers. In some cases, the bead may contain individual polymersthat may be further polymerized together. In some cases, beads may begenerated via polymerization of different precursors, such that theycomprise mixed polymers, co-polymers, and/or block co-polymers. In somecases, the bead may comprise covalent or ionic bonds between polymericprecursors (e.g., monomers, oligomers, linear polymers), nucleic acidmolecules (e.g., oligonucleotides), primers, and other entities. In somecases, the covalent bonds can be carbon-carbon bonds, thioether bonds,or carbon-heteroatom bonds.

Cross-linking may be permanent or reversible, depending upon theparticular cross-linker used. Reversible cross-linking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some cases, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides). Cystamine (including modified cystamines), forexample, is an organic agent comprising a disulfide bond that may beused as a crosslinker agent between individual monomeric or polymericprecursors of a bead. Polyacrylamide may be polymerized in the presenceof cystamine or a species comprising cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads comprising disulfidelinkages (e.g., chemically degradable beads comprisingchemically-reducible cross-linkers). The disulfide linkages may permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some cases, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more nucleic acid molecules (e.g.,barcode sequence, barcoded nucleic acid molecule, barcodedoligonucleotide, primer, or other oligonucleotide) to the bead. In somecases, an acrydite moiety can refer to an acrydite analogue generatedfrom the reaction of acrydite with one or more species, such as, thereaction of acrydite with other monomers and cross-linkers during apolymerization reaction. Acrydite moieties may be modified to formchemical bonds with a species to be attached, such as a nucleic acidmolecule (e.g., barcode sequence, barcoded nucleic acid molecule,barcoded oligonucleotide, primer, or other oligonucleotide). Acryditemoieties may be modified with thiol groups capable of forming adisulfide bond or may be modified with groups already comprising adisulfide bond. The thiol or disulfide (via disulfide exchange) may beused as an anchor point for a species to be attached or another part ofthe acrydite moiety may be used for attachment. In some cases,attachment can be reversible, such that when the disulfide bond isbroken (e.g., in the presence of a reducing agent), the attached speciesis released from the bead. In other cases, an acrydite moiety cancomprise a reactive hydroxyl group that may be used for attachment.

Functionalization of beads for attachment of nucleic acid molecules(e.g., oligonucleotides) may be achieved through a wide range ofdifferent approaches, including activation of chemical groups within apolymer, incorporation of active or activatable functional groups in thepolymer structure, or attachment at the pre-polymer or monomer stage inbead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g.,oligonucleotide), which may include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formessenger RNA) and/or one or more barcode sequences. The one morebarcode sequences may include sequences that are the same for allnucleic acid molecules coupled to a given bead and/or sequences that aredifferent across all nucleic acid molecules coupled to the given bead.The nucleic acid molecule may be incorporated into the bead.

In some cases, the nucleic acid molecule can comprise a functionalsequence, for example, for attachment to a sequencing flow cell, suchas, for example, a P5 sequence for Illumina® sequencing. In some cases,the nucleic acid molecule or derivative thereof (e.g., oligonucleotideor polynucleotide generated from the nucleic acid molecule) can compriseanother functional sequence, such as, for example, a P7 sequence forattachment to a sequencing flow cell for Illumina sequencing. In somecases, the nucleic acid molecule can comprise a barcode sequence. Insome cases, the primer can further comprise a unique molecularidentifier (UMI). In some cases, the primer can comprise an R1 primersequence for Illumina sequencing. In some cases, the primer can comprisean R2 primer sequence for Illumina sequencing. Examples of such nucleicacid molecules (e.g., oligonucleotides, polynucleotides, etc.) and usesthereof, as may be used with compositions, devices, methods and systemsof the present disclosure, are provided in U.S. Patent Pub. Nos.2014/0378345 and 2015/0376609, each of which is entirely incorporatedherein by reference.

FIG. 8 illustrates an example of a barcode carrying bead. A nucleic acidmolecule 802, such as an oligonucleotide, can be coupled to a bead 804by a releasable linkage 806, such as, for example, a disulfide linker.The same bead 804 may be coupled (e.g., via releasable linkage) to oneor more other nucleic acid molecules 818, 820. The nucleic acid molecule802 may be or comprise a barcode. As noted elsewhere herein, thestructure of the barcode may comprise a number of sequence elements. Thenucleic acid molecule 802 may comprise a functional sequence 808 thatmay be used in subsequent processing. For example, the functionalsequence 808 may include one or more of a sequencer specific flow cellattachment sequence (e.g., a P5 sequence for Illumina® sequencingsystems) and a sequencing primer sequence (e.g., a R1 primer forIllumina® sequencing systems). The nucleic acid molecule 802 maycomprise a barcode sequence 810 for use in barcoding the sample (e.g.,DNA, RNA, protein, etc.). In some cases, the barcode sequence 810 can bebead-specific such that the barcode sequence 810 is common to allnucleic acid molecules (e.g., including nucleic acid molecule 802)coupled to the same bead 804. Alternatively or in addition, the barcodesequence 810 can be partition-specific such that the barcode sequence810 is common to all nucleic acid molecules coupled to one or more beadsthat are partitioned into the same partition. The nucleic acid molecule802 may comprise a specific priming sequence 812, such as an mRNAspecific priming sequence (e.g., poly-T sequence), a targeted primingsequence, and/or a random priming sequence. The nucleic acid molecule802 may comprise an anchoring sequence 814 to ensure that the specificpriming sequence 812 hybridizes at the sequence end (e.g., of the mRNA).For example, the anchoring sequence 814 can include a random shortsequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longersequence, which can ensure that a poly-T segment is more likely tohybridize at the sequence end of the poly-A tail of the mRNA.

The nucleic acid molecule 802 may comprise a unique molecularidentifying sequence 816 (e.g., unique molecular identifier (UMI)). Insome cases, the unique molecular identifying sequence 816 may comprisefrom about 5 to about 8 nucleotides. Alternatively, the unique molecularidentifying sequence 816 may compress less than about 5 or more thanabout 8 nucleotides. The unique molecular identifying sequence 816 maybe a unique sequence that varies across individual nucleic acidmolecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g.,bead 804). In some cases, the unique molecular identifying sequence 816may be a random sequence (e.g., such as a random N-mer sequence). Forexample, the UMI may provide a unique identifier of the starting mRNAmolecule that was captured, in order to allow quantitation of the numberof original expressed RNA. As will be appreciated, although FIG. 8 showsthree nucleic acid molecules 802, 818, 820 coupled to the surface of thebead 804, an individual bead may be coupled to any number of individualnucleic acid molecules, for example, from one to tens to hundreds ofthousands or even millions of individual nucleic acid molecules. Therespective barcodes for the individual nucleic acid molecules cancomprise both common sequence segments or relatively common sequencesegments (e.g., 808, 810, 812, etc.) and variable or unique sequencesegments (e.g., 816) between different individual nucleic acid moleculescoupled to the same bead.

In operation, a biological particle (e.g., cell, DNA, RNA, etc.) can beco-partitioned along with a barcode bearing bead 804. The barcodednucleic acid molecules 802, 818, 820 can be released from the bead 804in the partition. By way of example, in the context of analyzing sampleRNA, the poly-T segment (e.g., 812) of one of the released nucleic acidmolecules (e.g., 802) can hybridize to the poly-A tail of an mRNAmolecule. Reverse transcription may result in a cDNA transcript of themRNA, but which transcript includes each of the sequence segments 808,810, 816 of the nucleic acid molecule 802. Because the nucleic acidmolecule 802 comprises an anchoring sequence 814, it will more likelyhybridize to and prime reverse transcription at the sequence end of thepoly-A tail of the mRNA. Within any given partition, all of the cDNAtranscripts of the individual mRNA molecules may include a commonbarcode sequence segment 810. However, the transcripts made from thedifferent mRNA molecules within a given partition may vary at the uniquemolecular identifying sequence 812 segment (e.g., UMI segment).Beneficially, even following any subsequent amplification of thecontents of a given partition, the number of different UMIs can beindicative of the quantity of mRNA originating from a given partition,and thus from the biological particle (e.g., cell). As noted above, thetranscripts can be amplified, cleaned up and sequenced to identify thesequence of the cDNA transcript of the mRNA, as well as to sequence thebarcode segment and the UMI segment. While a poly-T primer sequence isdescribed, other targeted or random priming sequences may also be usedin priming the reverse transcription reaction. Likewise, althoughdescribed as releasing the barcoded oligonucleotides into the partition,in some cases, the nucleic acid molecules bound to the bead (e.g., gelbead) may be used to hybridize and capture the mRNA on the solid phaseof the bead, for example, in order to facilitate the separation of theRNA from other cell contents.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such as N-ethylmaleimideor iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal to about 1:100,000,000,000, less than orequal to about 1:10,000,000,000, less than or equal to about1:1,000,000,000, less than or equal to about 1:100,000,000, less than orequal to about 1:10,000,000, less than or equal to about 1:1,000,000,less than or equal to about 1:100,000, less than or equal to about1:10,000) of reducing agent may be used for reduction. Controlling thenumber of disulfide linkages that are reduced to free thiols may beuseful in ensuring bead structural integrity during functionalization.In some cases, optically-active agents, such as fluorescent dyes may becoupled to beads via free thiol groups of the beads and used to quantifythe number of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

A bead injected or otherwise introduced into a partition may comprisereleasably, cleavably, or reversibly attached barcodes. A bead injectedor otherwise introduced into a partition may comprise activatablebarcodes. A bead injected or otherwise introduced into a partition maybe degradable, disruptable, or dissolvable beads.

Barcodes can be releasably, cleavably or reversibly attached to thebeads such that barcodes can be released or be releasable throughcleavage of a linkage between the barcode molecule and the bead, orreleased through degradation of the underlying bead itself, allowing thebarcodes to be accessed or be accessible by other reagents, or both. Innon-limiting examples, cleavage may be achieved through reduction ofdi-sulfide bonds, use of restriction enzymes, photo-activated cleavage,or cleavage via other types of stimuli (e.g., chemical, thermal, pH,enzymatic, etc.) and/or reactions, such as described elsewhere herein.Releasable barcodes may sometimes be referred to as being activatable,in that they are available for reaction once released. Thus, forexample, an activatable barcode may be activated by releasing thebarcode from a bead (or other suitable type of partition describedherein). Other activatable configurations are also envisioned in thecontext of the described methods and systems.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, such as barcode containingnucleic acid molecules (e.g., barcoded oligonucleotides), the beads maybe degradable, disruptable, or dissolvable spontaneously or uponexposure to one or more stimuli (e.g., temperature changes, pH changes,exposure to particular chemical species or phase, exposure to light,reducing agent, etc.). In some cases, a bead may be dissolvable, suchthat material components of the beads are solubilized when exposed to aparticular chemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead can be degradedor dissolved at elevated temperature and/or in basic conditions. In somecases, a bead may be thermally degradable such that when the bead isexposed to an appropriate change in temperature (e.g., heat), the beaddegrades. Degradation or dissolution of a bead bound to a species (e.g.,a nucleic acid molecule, e.g., barcoded oligonucleotide) may result inrelease of the species from the bead.

As will be appreciated from the above disclosure, the degradation of abead may refer to the disassociation of a bound or entrained speciesfrom a bead, both with and without structurally degrading the physicalbead itself. For example, the degradation of the bead may involvecleavage of a cleavable linkage via one or more species and/or methodsdescribed elsewhere herein. In another example, entrained species may bereleased from beads through osmotic pressure differences due to, forexample, changing chemical environments. By way of example, alterationof bead pore sizes due to osmotic pressure differences can generallyoccur without structural degradation of the bead itself. In some cases,an increase in pore size due to osmotic swelling of a bead can permitthe release of entrained species within the bead. In other cases,osmotic shrinking of a bead may cause a bead to better retain anentrained species due to pore size contraction.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., oligonucleotides, nucleic acid molecules) mayinteract with other reagents contained in the partition. For example, apolyacrylamide bead comprising cystamine and linked, via a disulfidebond, to a barcode sequence, may be combined with a reducing agentwithin a droplet of a water-in-oil emulsion. Within the droplet, thereducing agent can break the various disulfide bonds, resulting in beaddegradation and release of the barcode sequence into the aqueous, innerenvironment of the droplet. In another example, heating of a dropletcomprising a bead-bound barcode sequence in basic solution may alsoresult in bead degradation and release of the attached barcode sequenceinto the aqueous, inner environment of the droplet.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingnucleic acid molecule (e.g., oligonucleotide) bearing beads.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field. The swelling of the beads may beaccomplished by various swelling methods. The de-swelling of the beadsmay be accomplished, for instance, by transferring the beads in athermodynamically unfavorable solvent, subjecting the beads to lower orhigh temperatures, subjecting the beads to a lower or higher ionconcentration, and/or removing an electric field. The de-swelling of thebeads may be accomplished by various de-swelling methods. Transferringthe beads may cause pores in the bead to shrink. The shrinking may thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance may be due to steric interactions between thereagents and the interiors of the beads. The transfer may beaccomplished microfluidically. For instance, the transfer may beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore sizeof the beads may be adjusted by changing the polymer composition of thebead.

In some cases, an acrydite moiety linked to a precursor, another specieslinked to a precursor, or a precursor itself can comprise a labile bond,such as chemically, thermally, or photo-sensitive bond e.g., disulfidebond, UV sensitive bond, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the oligonucleotide, e.g., a barcode containing sequence, fromthe bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, enzymatic, etc.) such thatrelease of species attached to a bead via each labile bond may becontrolled by the application of the appropriate stimulus. Suchfunctionality may be useful in controlled release of species from a gelbead. In some cases, another species comprising a labile bond may belinked to a gel bead after gel bead formation via, for example, anactivated functional group of the gel bead as described above. As willbe appreciated, barcodes that are releasably, cleavably or reversiblyattached to the beads described herein include barcodes that arereleased or releasable through cleavage of a linkage between the barcodemolecule and the bead, or that are released through degradation of theunderlying bead itself, allowing the barcodes to be accessed oraccessible by other reagents, or both.

The barcodes that are releasable as described herein may sometimes bereferred to as being activatable, in that they are available forreaction once released. Thus, for example, an activatable barcode may beactivated by releasing the barcode from a bead (or other suitable typeof partition described herein). Other activatable configurations arealso envisioned in the context of the described methods and systems.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)). A bond may be cleavable via other nucleic acid moleculetargeting enzymes, such as restriction enzymes (e.g., restrictionendonucleases), as described further below.

Species may be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such species may or may notparticipate in polymerization. Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleicacid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,substrates, buffers), reagents for nucleic acid modification reactionssuch as polymerization, ligation, or digestion, and/or reagents fortemplate preparation (e.g., tagmentation) for one or more sequencingplatforms (e.g., Nextera® for Illumina®). Such species may include oneor more enzymes described herein, including without limitation,polymerase, reverse transcriptase, restriction enzymes (e.g.,endonuclease), transposase, ligase, proteinase K, DNAse, etc. Suchspecies may include one or more reagents described elsewhere herein(e.g., lysis agents, inhibitors, inactivating agents, chelating agents,stimulus). Trapping of such species may be controlled by the polymernetwork density generated during polymerization of precursors, controlof ionic charge within the gel bead (e.g., via ionic species linked topolymerized species), or by the release of other species. Encapsulatedspecies may be released from a bead upon bead degradation and/or byapplication of a stimulus capable of releasing the species from thebead. Alternatively or in addition, species may be partitioned in apartition (e.g., droplet) during or subsequent to partition formation.Such species may include, without limitation, the abovementioned speciesthat may also be encapsulated in a bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine. Upon exposure of the barcoded-bead to a reducingagent, the bead degrades and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, in many instances as noted above, thatdegradation may refer to the disassociation of a bound or entrainedspecies from a bead, both with and without structurally degrading thephysical bead itself. For example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself. In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

Where degradable beads are provided, it may be beneficial to avoidexposing such beads to the stimulus or stimuli that cause suchdegradation prior to a given time, in order to, for example, avoidpremature bead degradation and issues that arise from such degradation,including for example poor flow characteristics and aggregation. By wayof example, where beads comprise reducible cross-linking groups, such asdisulfide groups, it will be desirable to avoid contacting such beadswith reducing agents, e.g., DTT or other disulfide cleaving reagents. Insuch cases, treatment to the beads described herein will, in some casesbe provided free of reducing agents, such as DTT. Because reducingagents are often provided in commercial enzyme preparations, it may bedesirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that may be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than about 1/10th,less than about 1/50th, or even less than about 1/100th of the lowerranges for such materials used in degrading the beads. For example, forDTT, the reducing agent free preparation can have less than about 0.01millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even lessthan about 0.0001 mM DTT. In many cases, the amount of DTT can beundetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead. In some cases, any combination of stimulimay trigger degradation of a bead. For example, a change in pH mayenable a chemical agent (e.g., DTT) to become an effective reducingagent.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

Any suitable agent may degrade beads. In some embodiments, changes intemperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present ata concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, orgreater than 10 mM. The reducing agent may be present at concentrationof at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingoligonucleotide bearing beads.

Although FIG. 1 and FIG. 2 have been described in terms of providingsubstantially singly occupied partitions, above, in certain cases, itmay be desirable to provide multiply occupied partitions, e.g.,containing two, three, four or more cells and/or microcapsules (e.g.,beads) comprising barcoded nucleic acid molecules (e.g.,oligonucleotides) within a single partition. Accordingly, as notedabove, the flow characteristics of the biological particle and/or beadcontaining fluids and partitioning fluids may be controlled to providefor such multiply occupied partitions. In particular, the flowparameters may be controlled to provide a given occupancy rate atgreater than about 50% of the partitions, greater than about 75%, and insome cases greater than about 80%, 90%, 95%, or higher.

In some cases, additional microcapsules can be used to deliveradditional reagents to a partition. In such cases, it may beadvantageous to introduce different beads into a common channel ordroplet generation junction, from different bead sources (e.g.,containing different associated reagents) through different channelinlets into such common channel or droplet generation junction (e.g.,junction 210). In such cases, the flow and frequency of the differentbeads into the channel or junction may be controlled to provide for acertain ratio of microcapsules from each source, while ensuring a givenpairing or combination of such beads into a partition with a givennumber of biological particles (e.g., one biological particle and onebead per partition).

The partitions described herein may comprise small volumes, for example,less than about 10 microliters (μL), 5 μL, 14, 900 picoliters (pL), 800pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than about 1000 pL, 900 pL, 800 pL,700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10pL, 1 pL, or less. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedbiological particles and/or beads, within the partitions may be lessthan about 90% of the above described volumes, less than about 80%, lessthan about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, or less than about10% of the above described volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated or otherwise provided. Forexample, at least about 1,000 partitions, at least about 5,000partitions, at least about 10,000 partitions, at least about 50,000partitions, at least about 100,000 partitions, at least about 500,000partitions, at least about 1,000,000 partitions, at least about5,000,000 partitions at least about 10,000,000 partitions, at leastabout 50,000,000 partitions, at least about 100,000,000 partitions, atleast about 500,000,000 partitions, at least about 1,000,000,000partitions, or more partitions can be generated or otherwise provided.Moreover, the plurality of partitions may comprise both unoccupiedpartitions (e.g., empty partitions) and occupied partitions.

Reagents

In accordance with certain aspects, biological particles may bepartitioned along with lysis reagents in order to release the contentsof the biological particles within the partition. In such cases, thelysis agents can be contacted with the biological particle suspensionconcurrently with, or immediately prior to, the introduction of thebiological particles into the partitioning junction/droplet generationzone (e.g., junction 210), such as through an additional channel orchannels upstream of the channel junction. In accordance with otheraspects, additionally or alternatively, biological particles may bepartitioned along with other reagents, as will be described furtherbelow.

FIG. 3 shows an example of a microfluidic channel structure 300 forco-partitioning biological particles and reagents. The channel structure300 can include channel segments 301, 302, 304, 306 and 308. Channelsegments 301 and 302 communicate at a first channel junction 309.Channel segments 302, 304, 306, and 308 communicate at a second channeljunction 310.

In an example operation, the channel segment 301 may transport anaqueous fluid 312 that includes a plurality of biological particles 314along the channel segment 301 into the second junction 310. As analternative or in addition to, channel segment 301 may transport beads(e.g., gel beads). The beads may comprise barcode molecules.

For example, the channel segment 301 may be connected to a reservoircomprising an aqueous suspension of biological particles 314. Upstreamof, and immediately prior to reaching, the second junction 310, thechannel segment 301 may meet the channel segment 302 at the firstjunction 309. The channel segment 302 may transport a plurality ofreagents 315 (e.g., lysis agents) suspended in the aqueous fluid 312along the channel segment 302 into the first junction 309. For example,the channel segment 302 may be connected to a reservoir comprising thereagents 315. After the first junction 309, the aqueous fluid 312 in thechannel segment 301 can carry both the biological particles 314 and thereagents 315 towards the second junction 310. In some instances, theaqueous fluid 312 in the channel segment 301 can include one or morereagents, which can be the same or different reagents as the reagents315. A second fluid 316 that is immiscible with the aqueous fluid 312(e.g., oil) can be delivered to the second junction 310 from each ofchannel segments 304 and 306. Upon meeting of the aqueous fluid 312 fromthe channel segment 301 and the second fluid 316 from each of channelsegments 304 and 306 at the second channel junction 310, the aqueousfluid 312 can be partitioned as discrete droplets 318 in the secondfluid 316 and flow away from the second junction 310 along channelsegment 308. The channel segment 308 may deliver the discrete droplets318 to an outlet reservoir fluidly coupled to the channel segment 308,where they may be harvested.

The second fluid 316 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets318.

A discrete droplet generated may include an individual biologicalparticle 314 and/or one or more reagents 315. In some instances, adiscrete droplet generated may include a barcode carrying bead (notshown), such as via other microfluidics structures described elsewhereherein. In some instances, a discrete droplet may be unoccupied (e.g.,no reagents, no biological particles).

Beneficially, when lysis reagents and biological particles areco-partitioned, the lysis reagents can facilitate the release of thecontents of the biological particles within the partition. The contentsreleased in a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 300 may have other geometries. For example, amicrofluidic channel structure can have more than two channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, 5channel segments or more each carrying the same or different types ofbeads, reagents, and/or biological particles that meet at a channeljunction. Fluid flow in each channel segment may be controlled tocontrol the partitioning of the different elements into droplets. Fluidmay be directed flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

Examples of lysis agents include bioactive reagents, such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, etc., such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other lysis enzymes available from, e.g.,Sigma-Aldrich, Inc. (St Louis, MO), as well as other commerciallyavailable lysis enzymes. Other lysis agents may additionally oralternatively be co-partitioned with the biological particles to causethe release of the biological particles's contents into the partitions.For example, in some cases, surfactant-based lysis solutions may be usedto lyse cells, although these may be less desirable for emulsion basedsystems where the surfactants can interfere with stable emulsions. Insome cases, lysis solutions may include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some cases, lysis solutionsmay include ionic surfactants such as, for example, sarcosyl and sodiumdodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanicalcellular disruption may also be used in certain cases, e.g.,non-emulsion based partitioning such as encapsulation of biologicalparticles that may be in addition to or in place of dropletpartitioning, where any pore size of the encapsulate is sufficientlysmall to retain nucleic acid fragments of a given size, followingcellular disruption.

Alternatively or in addition to the lysis agents co-partitioned with thebiological particles described above, other reagents can also beco-partitioned with the biological particles, including, for example,DNase and RNase inactivating agents or inhibitors, such as proteinase K,chelating agents, such as EDTA, and other reagents employed in removingor otherwise reducing negative activity or impact of different celllysate components on subsequent processing of nucleic acids. Inaddition, in the case of encapsulated biological particles, thebiological particles may be exposed to an appropriate stimulus torelease the biological particles or their contents from a co-partitionedmicrocapsule. For example, in some cases, a chemical stimulus may beco-partitioned along with an encapsulated biological particle to allowfor the degradation of the microcapsule and release of the cell or itscontents into the larger partition. In some cases, this stimulus may bethe same as the stimulus described elsewhere herein for release ofnucleic acid molecules (e.g., oligonucleotides) from their respectivemicrocapsule (e.g., bead). In alternative aspects, this may be adifferent and non-overlapping stimulus, in order to allow anencapsulated biological particle to be released into a partition at adifferent time from the release of nucleic acid molecules into the samepartition.

Additional reagents may also be co-partitioned with the biologicalparticles, such as endonucleases to fragment a biological particle'sDNA, DNA polymerase enzymes and dNTPs used to amplify the biologicalparticle's nucleic acid fragments and to attach the barcode moleculartags to the amplified fragments. Other enzymes may be co-partitioned,including without limitation, polymerase, transposase, ligase,proteinase K, DNAse, etc. Additional reagents may also include reversetranscriptase enzymes, including enzymes with terminal transferaseactivity, primers and oligonucleotides, and switch oligonucleotides(also referred to herein as “switch oligos” or “template switchingoligonucleotides”) which can be used for template switching. In somecases, template switching can be used to increase the length of a cDNA.In some cases, template switching can be used to append a predefinednucleic acid sequence to the cDNA. In an example of template switching,cDNA can be generated from reverse transcription of a template, e.g.,cellular mRNA, where a reverse transcriptase with terminal transferaseactivity can add additional nucleotides, e.g., polyC, to the cDNA in atemplate independent manner. Switch oligos can include sequencescomplementary to the additional nucleotides, e.g., polyG. The additionalnucleotides (e.g., polyC) on the cDNA can hybridize to the additionalnucleotides (e.g., polyG) on the switch oligo, whereby the switch oligocan be used by the reverse transcriptase as template to further extendthe cDNA. Template switching oligonucleotides may comprise ahybridization region and a template region. The hybridization region cancomprise any sequence capable of hybridizing to the target. In somecases, as previously described, the hybridization region comprises aseries of G bases to complement the overhanging C bases at the 3′ end ofa cDNA molecule. The series of G bases may comprise 1 G base, 2 G bases,3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The templatesequence can comprise any sequence to be incorporated into the cDNA. Insome cases, the template region comprises at least 1 (e.g., at least 2,3, 4, 5 or more) tag sequences and/or functional sequences. Switcholigos may comprise deoxyribonucleic acids; ribonucleic acids; modifiednucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA),inverted dT, 5-Methyl dC, 2′-deoxylnosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination.

In some cases, the length of a switch oligo may be at least about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides or longer.

In some cases, the length of a switch oligo may be at most about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides.

Once the contents of the cells are released into their respectivepartitions, the macromolecular components (e.g., macromolecularconstituents of biological particles, such as RNA, DNA, or proteins)contained therein may be further processed within the partitions. Inaccordance with the methods and systems described herein, themacromolecular component contents of individual biological particles canbe provided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same biological particle or particles. The ability toattribute characteristics to individual biological particles or groupsof biological particles is provided by the assignment of uniqueidentifiers specifically to an individual biological particle or groupsof biological particles. Unique identifiers, e.g., in the form ofnucleic acid barcodes can be assigned or associated with individualbiological particles or populations of biological particles, in order totag or label the biological particle's macromolecular components (and asa result, its characteristics) with the unique identifiers. These uniqueidentifiers can then be used to attribute the biological particle'scomponents and characteristics to an individual biological particle orgroup of biological particles.

In some aspects, this is performed by co-partitioning the individualbiological particle or groups of biological particles with the uniqueidentifiers, such as described above (with reference to FIG. 2 ). Insome aspects, the unique identifiers are provided in the form of nucleicacid molecules (e.g., oligonucleotides) that comprise nucleic acidbarcode sequences that may be attached to or otherwise associated withthe nucleic acid contents of individual biological particle, or to othercomponents of the biological particle, and particularly to fragments ofthose nucleic acids. The nucleic acid molecules are partitioned suchthat as between nucleic acid molecules in a given partition, the nucleicacid barcode sequences contained therein are the same, but as betweendifferent partitions, the nucleic acid molecule can, and do havediffering barcode sequences, or at least represent a large number ofdifferent barcode sequences across all of the partitions in a givenanalysis. In some aspects, only one nucleic acid barcode sequence can beassociated with a given partition, although in some cases, two or moredifferent barcode sequences may be present.

The nucleic acid barcode sequences can include from about 6 to about 20or more nucleotides within the sequence of the nucleic acid molecules(e.g., oligonucleotides). The nucleic acid barcode sequences can includefrom about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides. In some cases, the length of a barcode sequence may beabout 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotidesor longer. In some cases, the length of a barcode sequence may be atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20nucleotides or longer. In some cases, the length of a barcode sequencemay be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 nucleotides or shorter. These nucleotides may be completelycontiguous, i.e., in a single stretch of adjacent nucleotides, or theymay be separated into two or more separate subsequences that areseparated by 1 or more nucleotides. In some cases, separated barcodesubsequences can be from about 4 to about 16 nucleotides in length. Insome cases, the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or longer. In some cases, the barcode subsequence maybe at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned nucleic acid molecules can also comprise otherfunctional sequences useful in the processing of the nucleic acids fromthe co-partitioned biological particles. These sequences include, e.g.,targeted or random/universal amplification primer sequences foramplifying the genomic DNA from the individual biological particleswithin the partitions while attaching the associated barcode sequences,sequencing primers or primer recognition sites, hybridization or probingsequences, e.g., for identification of presence of the sequences or forpulling down barcoded nucleic acids, or any of a number of otherpotential functional sequences. Other mechanisms of co-partitioningoligonucleotides may also be employed, including, e.g., coalescence oftwo or more droplets, where one droplet contains oligonucleotides, ormicrodispensing of oligonucleotides into partitions, e.g., dropletswithin microfluidic systems.

In an example, microcapsules, such as beads, are provided that eachinclude large numbers of the above described barcoded nucleic acidmolecules (e.g., barcoded oligonucleotides) releasably attached to thebeads, where all of the nucleic acid molecules attached to a particularbead will include the same nucleic acid barcode sequence, but where alarge number of diverse barcode sequences are represented across thepopulation of beads used. In some embodiments, hydrogel beads, e.g.,comprising polyacrylamide polymer matrices, are used as a solid supportand delivery vehicle for the nucleic acid molecules into the partitions,as they are capable of carrying large numbers of nucleic acid molecules,and may be configured to release those nucleic acid molecules uponexposure to a particular stimulus, as described elsewhere herein. Insome cases, the population of beads provides a diverse barcode sequencelibrary that includes at least about 1,000 different barcode sequences,at least about 5,000 different barcode sequences, at least about 10,000different barcode sequences, at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences, or more. Additionally, each bead can be provided withlarge numbers of nucleic acid (e.g., oligonucleotide) moleculesattached. In particular, the number of molecules of nucleic acidmolecules including the barcode sequence on an individual bead can be atleast about 1,000 nucleic acid molecules, at least about 5,000 nucleicacid molecules, at least about 10,000 nucleic acid molecules, at leastabout 50,000 nucleic acid molecules, at least about 100,000 nucleic acidmolecules, at least about 500,000 nucleic acids, at least about1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acidmolecules, at least about 10,000,000 nucleic acid molecules, at leastabout 50,000,000 nucleic acid molecules, at least about 100,000,000nucleic acid molecules, at least about 250,000,000 nucleic acidmolecules and in some cases at least about 1 billion nucleic acidmolecules, or more. Nucleic acid molecules of a given bead can includeidentical (or common) barcode sequences, different barcode sequences, ora combination of both. Nucleic acid molecules of a given bead caninclude multiple sets of nucleic acid molecules. Nucleic acid moleculesof a given set can include identical barcode sequences. The identicalbarcode sequences can be different from barcode sequences of nucleicacid molecules of another set.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences. Additionally, each partition of the population caninclude at least about 1,000 nucleic acid molecules, at least about5,000 nucleic acid molecules, at least about 10,000 nucleic acidmolecules, at least about 50,000 nucleic acid molecules, at least about100,000 nucleic acid molecules, at least about 500,000 nucleic acids, atleast about 1,000,000 nucleic acid molecules, at least about 5,000,000nucleic acid molecules, at least about 10,000,000 nucleic acidmolecules, at least about 50,000,000 nucleic acid molecules, at leastabout 100,000,000 nucleic acid molecules, at least about 250,000,000nucleic acid molecules and in some cases at least about 1 billionnucleic acid molecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known set of barcode sequences may provide greater assuranceof identification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The nucleic acid molecules (e.g., oligonucleotides) are releasable fromthe beads upon the application of a particular stimulus to the beads. Insome cases, the stimulus may be a photo-stimulus, e.g., through cleavageof a photo-labile linkage that releases the nucleic acid molecules. Inother cases, a thermal stimulus may be used, where elevation of thetemperature of the beads environment will result in cleavage of alinkage or other release of the nucleic acid molecules form the beads.In still other cases, a chemical stimulus can be used that cleaves alinkage of the nucleic acid molecules to the beads, or otherwise resultsin release of the nucleic acid molecules from the beads. In one case,such compositions include the polyacrylamide matrices described abovefor encapsulation of biological particles, and may be degraded forrelease of the attached nucleic acid molecules through exposure to areducing agent, such as DTT.

In some aspects, provided are systems and methods for controlledpartitioning. Droplet size may be controlled by adjusting certaingeometric features in channel architecture (e.g., microfluidics channelarchitecture). For example, an expansion angle, width, and/or length ofa channel may be adjusted to control droplet size.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets. A channelstructure 400 can include a channel segment 402 communicating at achannel junction 406 (or intersection) with a reservoir 404. Thereservoir 404 can be a chamber. Any reference to “reservoir,” as usedherein, can also refer to a “chamber.” In operation, an aqueous fluid408 that includes suspended beads 412 may be transported along thechannel segment 402 into the junction 406 to meet a second fluid 410that is immiscible with the aqueous fluid 408 in the reservoir 404 tocreate droplets 416, 418 of the aqueous fluid 408 flowing into thereservoir 404. At the junction 406 where the aqueous fluid 408 and thesecond fluid 410 meet, droplets can form based on factors such as thehydrodynamic forces at the junction 406, flow rates of the two fluids408, 410, fluid properties, and certain geometric parameters (e.g., w,h₀, α, etc.) of the channel structure 400. A plurality of droplets canbe collected in the reservoir 404 by continuously injecting the aqueousfluid 408 from the channel segment 402 through the junction 406.

A discrete droplet generated may include a bead (e.g., as in occupieddroplets 416). Alternatively, a discrete droplet generated may includemore than one bead. Alternatively, a discrete droplet generated may notinclude any beads (e.g., as in unoccupied droplet 418). In someinstances, a discrete droplet generated may contain one or morebiological particles, as described elsewhere herein. In some instances,a discrete droplet generated may comprise one or more reagents, asdescribed elsewhere herein.

In some instances, the aqueous fluid 408 can have a substantiallyuniform concentration or frequency of beads 412. The beads 412 can beintroduced into the channel segment 402 from a separate channel (notshown in FIG. 4 ). The frequency of beads 412 in the channel segment 402may be controlled by controlling the frequency in which the beads 412are introduced into the channel segment 402 and/or the relative flowrates of the fluids in the channel segment 402 and the separate channel.In some instances, the beads can be introduced into the channel segment402 from a plurality of different channels, and the frequency controlledaccordingly.

In some instances, the aqueous fluid 408 in the channel segment 402 cancomprise biological particles (e.g., described with reference to FIGS. 1and 2 ). In some instances, the aqueous fluid 408 can have asubstantially uniform concentration or frequency of biologicalparticles. As with the beads, the biological particles can be introducedinto the channel segment 402 from a separate channel. The frequency orconcentration of the biological particles in the aqueous fluid 408 inthe channel segment 402 may be controlled by controlling the frequencyin which the biological particles are introduced into the channelsegment 402 and/or the relative flow rates of the fluids in the channelsegment 402 and the separate channel. In some instances, the biologicalparticles can be introduced into the channel segment 402 from aplurality of different channels, and the frequency controlledaccordingly. In some instances, a first separate channel can introducebeads and a second separate channel can introduce biological particlesinto the channel segment 402. The first separate channel introducing thebeads may be upstream or downstream of the second separate channelintroducing the biological particles.

The second fluid 410 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resultingdroplets.

In some instances, the second fluid 410 may not be subjected to and/ordirected to any flow in or out of the reservoir 404. For example, thesecond fluid 410 may be substantially stationary in the reservoir 404.In some instances, the second fluid 410 may be subjected to flow withinthe reservoir 404, but not in or out of the reservoir 404, such as viaapplication of pressure to the reservoir 404 and/or as affected by theincoming flow of the aqueous fluid 408 at the junction 406.Alternatively, the second fluid 410 may be subjected and/or directed toflow in or out of the reservoir 404. For example, the reservoir 404 canbe a channel directing the second fluid 410 from upstream to downstream,transporting the generated droplets.

The channel structure 400 at or near the junction 406 may have certaingeometric features that at least partly determine the sizes of thedroplets formed by the channel structure 400. The channel segment 402can have a height, h₀ and width, w, at or near the junction 406. By wayof example, the channel segment 402 can comprise a rectangularcross-section that leads to a reservoir 404 having a wider cross-section(such as in width or diameter). Alternatively, the cross-section of thechannel segment 402 can be other shapes, such as a circular shape,trapezoidal shape, polygonal shape, or any other shapes. The top andbottom walls of the reservoir 404 at or near the junction 406 can beinclined at an expansion angle, a. The expansion angle, a, allows thetongue (portion of the aqueous fluid 408 leaving channel segment 402 atjunction 406 and entering the reservoir 404 before droplet formation) toincrease in depth and facilitate decrease in curvature of theintermediately formed droplet. Droplet size may decrease with increasingexpansion angle. The resulting droplet radius, R_(d), may be predictedby the following equation for the aforementioned geometric parameters ofh₀, w, and α:

$R_{d} \approx {0.44\left( {1 + {2.2\sqrt{\tan\tan\alpha}\frac{w}{h_{0}}}} \right)\frac{h_{0}}{\sqrt{\tan\tan\alpha}}}$

By way of example, for a channel structure with w=21 μm, h=21 μm, andα=3°, the predicted droplet size is 121 μm. In another example, for achannel structure with w=25 h=25 μm, and α=5°, the predicted dropletsize is 123 μm. In another example, for a channel structure with w=28μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm.

In some instances, the expansion angle, a, may be between a range offrom about 0.5° to about 4°, from about 0.1° to about 10°, or from about0° to about 90°. For example, the expansion angle can be at least about0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°,4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°,55°, 60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, theexpansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°,82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°,20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.In some instances, the width, w, can be between a range of from about100 micrometers (μm) to about 500 μm. In some instances, the width, w,can be between a range of from about 10 μm to about 200 μm.Alternatively, the width can be less than about 10 μm. Alternatively,the width can be greater than about 500 μm. In some instances, the flowrate of the aqueous fluid 408 entering the junction 406 can be betweenabout 0.04 microliters (μL)/minute (min) and about 40 μL/min. In someinstances, the flow rate of the aqueous fluid 408 entering the junction406 can be between about 0.01 microliters (μL)/minute (min) and about100 μL/min. Alternatively, the flow rate of the aqueous fluid 408entering the junction 406 can be less than about 0.01 μL/min.Alternatively, the flow rate of the aqueous fluid 408 entering thejunction 406 can be greater than about 40 μL/min, such as 45 μL/min, 50μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75 μL/min, 80μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110 μL/min, 120μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. At lower flowrates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 408 entering the junction 406.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

The throughput of droplet generation can be increased by increasing thepoints of generation, such as increasing the number of junctions (e.g.,junction 406) between aqueous fluid 408 channel segments (e.g., channelsegment 402) and the reservoir 404. Alternatively or in addition, thethroughput of droplet generation can be increased by increasing the flowrate of the aqueous fluid 408 in the channel segment 402.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 500 can comprise a plurality of channel segments 502 and areservoir 504. Each of the plurality of channel segments 502 may be influid communication with the reservoir 504. The channel structure 500can comprise a plurality of channel junctions 506 between the pluralityof channel segments 502 and the reservoir 504. Each channel junction canbe a point of droplet generation. The channel segment 402 from thechannel structure 400 in FIG. 4 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 502 in channel structure 500 and any description to thecorresponding components thereof. The reservoir 404 from the channelstructure 400 and any description to the components thereof maycorrespond to the reservoir 504 from the channel structure 500 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 502 maycomprise an aqueous fluid 508 that includes suspended beads 512. Thereservoir 504 may comprise a second fluid 510 that is immiscible withthe aqueous fluid 508. In some instances, the second fluid 510 may notbe subjected to and/or directed to any flow in or out of the reservoir504. For example, the second fluid 510 may be substantially stationaryin the reservoir 504. In some instances, the second fluid 510 may besubjected to flow within the reservoir 504, but not in or out of thereservoir 504, such as via application of pressure to the reservoir 504and/or as affected by the incoming flow of the aqueous fluid 508 at thejunctions. Alternatively, the second fluid 510 may be subjected and/ordirected to flow in or out of the reservoir 504. For example, thereservoir 504 can be a channel directing the second fluid 510 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 508 that includes suspended beads 512may be transported along the plurality of channel segments 502 into theplurality of junctions 506 to meet the second fluid 510 in the reservoir504 to create droplets 516, 518. A droplet may form from each channelsegment at each corresponding junction with the reservoir 504. At thejunction where the aqueous fluid 508 and the second fluid 510 meet,droplets can form based on factors such as the hydrodynamic forces atthe junction, flow rates of the two fluids 508, 510, fluid properties,and certain geometric parameters (e.g., w, h₀, α, etc.) of the channelstructure 500, as described elsewhere herein. A plurality of dropletscan be collected in the reservoir 504 by continuously injecting theaqueous fluid 508 from the plurality of channel segments 502 through theplurality of junctions 506. Throughput may significantly increase withthe parallel channel configuration of channel structure 500. Forexample, a channel structure having five inlet channel segmentscomprising the aqueous fluid 508 may generate droplets five times asfrequently than a channel structure having one inlet channel segment,provided that the fluid flow rate in the channel segments aresubstantially the same. The fluid flow rate in the different inletchannel segments may or may not be substantially the same. A channelstructure may have as many parallel channel segments as is practical andallowed for the size of the reservoir. For example, the channelstructure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantiallyparallel channel segments.

The geometric parameters, w, h₀, and α, may or may not be uniform foreach of the channel segments in the plurality of channel segments 502.For example, each channel segment may have the same or different widthsat or near its respective channel junction with the reservoir 504. Forexample, each channel segment may have the same or different height ator near its respective channel junction with the reservoir 504. Inanother example, the reservoir 504 may have the same or differentexpansion angle at the different channel junctions with the plurality ofchannel segments 502. When the geometric parameters are uniform,beneficially, droplet size may also be controlled to be uniform evenwith the increased throughput. In some instances, when it is desirableto have a different distribution of droplet sizes, the geometricparameters for the plurality of channel segments 502 may be variedaccordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 600 can comprise a plurality of channel segments 602 arrangedgenerally circularly around the perimeter of a reservoir 604. Each ofthe plurality of channel segments 602 may be in fluid communication withthe reservoir 604. The channel structure 600 can comprise a plurality ofchannel junctions 606 between the plurality of channel segments 602 andthe reservoir 604. Each channel junction can be a point of dropletgeneration. The channel segment 402 from the channel structure 400 inFIG. 2 and any description to the components thereof may correspond to agiven channel segment of the plurality of channel segments 602 inchannel structure 600 and any description to the correspondingcomponents thereof. The reservoir 404 from the channel structure 400 andany description to the components thereof may correspond to thereservoir 604 from the channel structure 600 and any description to thecorresponding components thereof.

Each channel segment of the plurality of channel segments 602 maycomprise an aqueous fluid 608 that includes suspended beads 612. Thereservoir 604 may comprise a second fluid 610 that is immiscible withthe aqueous fluid 608. In some instances, the second fluid 610 may notbe subjected to and/or directed to any flow in or out of the reservoir604. For example, the second fluid 610 may be substantially stationaryin the reservoir 604. In some instances, the second fluid 610 may besubjected to flow within the reservoir 604, but not in or out of thereservoir 604, such as via application of pressure to the reservoir 604and/or as affected by the incoming flow of the aqueous fluid 608 at thejunctions. Alternatively, the second fluid 610 may be subjected and/ordirected to flow in or out of the reservoir 604. For example, thereservoir 604 can be a channel directing the second fluid 610 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 608 that includes suspended beads 612may be transported along the plurality of channel segments 602 into theplurality of junctions 606 to meet the second fluid 610 in the reservoir604 to create a plurality of droplets 616. A droplet may form from eachchannel segment at each corresponding junction with the reservoir 604.At the junction where the aqueous fluid 608 and the second fluid 610meet, droplets can form based on factors such as the hydrodynamic forcesat the junction, flow rates of the two fluids 608, 610, fluidproperties, and certain geometric parameters (e.g., widths and heightsof the channel segments 602, expansion angle of the reservoir 604, etc.)of the channel structure 600, as described elsewhere herein. A pluralityof droplets can be collected in the reservoir 604 by continuouslyinjecting the aqueous fluid 608 from the plurality of channel segments602 through the plurality of junctions 606. Throughput may significantlyincrease with the substantially parallel channel configuration of thechannel structure 600. A channel structure may have as manysubstantially parallel channel segments as is practical and allowed forby the size of the reservoir. For example, the channel structure mayhave at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 1500, 5000 or more parallel or substantially parallel channelsegments. The plurality of channel segments may be substantially evenlyspaced apart, for example, around an edge or perimeter of the reservoir.Alternatively, the spacing of the plurality of channel segments may beuneven.

The reservoir 604 may have an expansion angle, a (not shown in FIG. 6 )at or near each channel junction. Each channel segment of the pluralityof channel segments 602 may have a width, w, and a height, h₀, at ornear the channel junction. The geometric parameters, w, h₀, and α, mayor may not be uniform for each of the channel segments in the pluralityof channel segments 602. For example, each channel segment may have thesame or different widths at or near its respective channel junction withthe reservoir 604. For example, each channel segment may have the sameor different height at or near its respective channel junction with thereservoir 604.

The reservoir 604 may have the same or different expansion angle at thedifferent channel junctions with the plurality of channel segments 602.For example, a circular reservoir (as shown in FIG. 6 ) may have aconical, dome-like, or hemispherical ceiling (e.g., top wall) to providethe same or substantially same expansion angle for each channel segments602 at or near the plurality of channel junctions 606. When thegeometric parameters are uniform, beneficially, resulting droplet sizemay be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channelsegments 602 may be varied accordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size. The beads and/orbiological particle injected into the droplets may or may not haveuniform size.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.A channel structure 700 can include a channel segment 702 communicatingat a channel junction 706 (or intersection) with a reservoir 704. Insome instances, the channel structure 700 and one or more of itscomponents can correspond to the channel structure 100 and one or moreof its components. FIG. 7B shows a perspective view of the channelstructure 700 of FIG. 7A.

An aqueous fluid 712 comprising a plurality of particles 716 may betransported along the channel segment 702 into the junction 706 to meeta second fluid 714 (e.g., oil, etc.) that is immiscible with the aqueousfluid 712 in the reservoir 704 to create droplets 720 of the aqueousfluid 712 flowing into the reservoir 704. At the junction 706 where theaqueous fluid 712 and the second fluid 714 meet, droplets can form basedon factors such as the hydrodynamic forces at the junction 706, relativeflow rates of the two fluids 712, 714, fluid properties, and certaingeometric parameters (e.g., Δh, etc.) of the channel structure 700. Aplurality of droplets can be collected in the reservoir 704 bycontinuously injecting the aqueous fluid 712 from the channel segment702 at the junction 706.

A discrete droplet generated may comprise one or more particles of theplurality of particles 716. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, biologicalparticle, macromolecular constituents of biological particle, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 712 can have a substantiallyuniform concentration or frequency of particles 716. As describedelsewhere herein (e.g., with reference to FIG. 4 ), the particles 716(e.g., beads) can be introduced into the channel segment 702 from aseparate channel (not shown in FIG. 7 ). The frequency of particles 716in the channel segment 702 may be controlled by controlling thefrequency in which the particles 716 are introduced into the channelsegment 702 and/or the relative flow rates of the fluids in the channelsegment 702 and the separate channel. In some instances, the particles716 can be introduced into the channel segment 702 from a plurality ofdifferent channels, and the frequency controlled accordingly. In someinstances, different particles may be introduced via separate channels.For example, a first separate channel can introduce beads and a secondseparate channel can introduce biological particles into the channelsegment 702. The first separate channel introducing the beads may beupstream or downstream of the second separate channel introducing thebiological particles.

In some instances, the second fluid 714 may not be subjected to and/ordirected to any flow in or out of the reservoir 704. For example, thesecond fluid 714 may be substantially stationary in the reservoir 704.In some instances, the second fluid 714 may be subjected to flow withinthe reservoir 704, but not in or out of the reservoir 704, such as viaapplication of pressure to the reservoir 704 and/or as affected by theincoming flow of the aqueous fluid 712 at the junction 706.Alternatively, the second fluid 714 may be subjected and/or directed toflow in or out of the reservoir 704. For example, the reservoir 704 canbe a channel directing the second fluid 714 from upstream to downstream,transporting the generated droplets.

The channel structure 700 at or near the junction 706 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the channel structure 700. The channelsegment 702 can have a first cross-section height, h₁, and the reservoir704 can have a second cross-section height, h₂. The first cross-sectionheight, h₁, and the second cross-section height, h₂, may be different,such that at the junction 706, there is a height difference of Δh. Thesecond cross-section height, h₂, may be greater than the firstcross-section height, h₁. In some instances, the reservoir maythereafter gradually increase in cross-section height, for example, themore distant it is from the junction 706. In some instances, thecross-section height of the reservoir may increase in accordance withexpansion angle, β, at or near the junction 706. The height difference,Δh, and/or expansion angle, β, can allow the tongue (portion of theaqueous fluid 712 leaving channel segment 702 at junction 706 andentering the reservoir 704 before droplet formation) to increase indepth and facilitate decrease in curvature of the intermediately formeddroplet. For example, droplet size may decrease with increasing heightdifference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 μm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 μm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less. In some instances, theexpansion angle, β, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 712 entering thejunction 706 can be between about 0.04 microliters (μL)/minute (min) andabout 40 μL/min. In some instances, the flow rate of the aqueous fluid712 entering the junction 706 can be between about 0.01 microliters(μL)/minute (min) and about 100 μL/min. Alternatively, the flow rate ofthe aqueous fluid 712 entering the junction 706 can be less than about0.01 μL/min. Alternatively, the flow rate of the aqueous fluid 712entering the junction 706 can be greater than about 40 μL/min, such as45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110μL/min, 120 μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. Atlower flow rates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 712 entering the junction 706. The secondfluid 714 may be stationary, or substantially stationary, in thereservoir 704. Alternatively, the second fluid 714 may be flowing, suchas at the above flow rates described for the aqueous fluid 712.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

While FIGS. 7A and 7B illustrate the height difference, Δh, being abruptat the junction 706 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 μm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 706, a bottom wall of thechannel and a bottom wall of the reservoir can meet at an angle greaterthan 90°. Alternatively or in addition, a top wall (e.g., ceiling) ofthe channel and a top wall (e.g., ceiling) of the reservoir can meet anangle greater than 90°. A gradual increase or decrease may be linear ornon-linear (e.g., exponential, sinusoidal, etc.). Alternatively or inaddition, the height difference may variably increase and/or decreaselinearly or non-linearly. While FIGS. 7A and 7B illustrate the expandingreservoir cross-section height as linear (e.g., constant expansionangle, β), the cross-section height may expand non-linearly. Forexample, the reservoir may be defined at least partially by a dome-like(e.g., hemispherical) shape having variable expansion angles. Thecross-section height may expand in any shape.

The channel networks, e.g., as described above or elsewhere herein, canbe fluidly coupled to appropriate fluidic components. For example, theinlet channel segments are fluidly coupled to appropriate sources of thematerials they are to deliver to a channel junction. These sources mayinclude any of a variety of different fluidic components, from simplereservoirs defined in or connected to a body structure of a microfluidicdevice, to fluid conduits that deliver fluids from off-device sources,manifolds, fluid flow units (e.g., actuators, pumps, compressors) or thelike. Likewise, the outlet channel segment (e.g., channel segment 208,reservoir 604, etc.) may be fluidly coupled to a receiving vessel orconduit for the partitioned cells for subsequent processing. Again, thismay be a reservoir defined in the body of a microfluidic device, or itmay be a fluidic conduit for delivering the partitioned cells to asubsequent process operation, instrument or component.

The methods and systems described herein may be used to greatly increasethe efficiency of single cell applications and/or other applicationsreceiving droplet-based input. For example, following the sorting ofoccupied cells and/or appropriately-sized cells, subsequent operationsthat can be performed can include generation of amplification products,purification (e.g., via solid phase reversible immobilization (SPRI)),further processing (e.g., shearing, ligation of functional sequences,and subsequent amplification (e.g., via PCR)). These operations mayoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled for additional operations. Additionalreagents that may be co-partitioned along with the barcode bearing beadmay include oligonucleotides to block ribosomal RNA (rRNA) and nucleasesto digest genomic DNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing and/or sequence the5′ end of a polynucleotide sequence. The amplification products, forexample, first amplification products and/or second amplificationproducts, may be subject to sequencing for sequence analysis. In somecases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method.

A variety of applications require the evaluation of the presence andquantification of different biological particle or organism types withina population of biological particles, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

Computer Systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 23 shows a computer system2301 that is programmed or otherwise configured to, for example, (i)control a microfluidics system (e.g., fluid flow), (ii) sort occupieddroplets from unoccupied droplets, (iii) polymerize droplets, (iv)perform sequencing applications, or (v) generate and maintain a libraryof nucleic acid molecules. The computer system 2301 can regulate variousaspects of the present disclosure, such as, for example, fluid flowrates in one or more channels in a microfluidic structure,polymerization application units, etc. The computer system 2301 can bean electronic device of a user or a computer system that is remotelylocated with respect to the electronic device. The electronic device canbe a mobile electronic device.

The computer system 2301 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 2305, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 2301 also includes memory or memorylocation 2310 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 2315 (e.g., hard disk), communicationinterface 2320 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 2325, such as cache, othermemory, data storage and/or electronic display adapters. The memory2310, storage unit 2315, interface 2320 and peripheral devices 2325 arein communication with the CPU 2305 through a communication bus (solidlines), such as a motherboard. The storage unit 2315 can be a datastorage unit (or data repository) for storing data. The computer system2301 can be operatively coupled to a computer network (“network”) 2330with the aid of the communication interface 2320. The network 2330 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 2330 insome cases is a telecommunication and/or data network. The network 2330can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 2330, in some cases withthe aid of the computer system 2301, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 2301 tobehave as a client or a server.

The CPU 2305 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 2310. The instructionscan be directed to the CPU 2305, which can subsequently program orotherwise configure the CPU 2305 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 2305 can includefetch, decode, execute, and writeback.

The CPU 2305 can be part of a circuit, such as an integrated circuit.One or more other components of the system 2301 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 2315 can store files, such as drivers, libraries andsaved programs. The storage unit 2315 can store user data, e.g., userpreferences and user programs. The computer system 2301 in some casescan include one or more additional data storage units that are externalto the computer system 2301, such as located on a remote server that isin communication with the computer system 2301 through an intranet orthe Internet.

The computer system 2301 can communicate with one or more remotecomputer systems through the network 2330. For instance, the computersystem 2301 can communicate with a remote computer system of a user(e.g., operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 2301 via the network 2330.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 2301, such as, for example, on thememory 2310 or electronic storage unit 2315. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 2305. In some cases, thecode can be retrieved from the storage unit 2315 and stored on thememory 2310 for ready access by the processor 2305. In some situations,the electronic storage unit 2315 can be precluded, andmachine-executable instructions are stored on memory 2310.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 2301, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 2301 can include or be in communication with anelectronic display 2335 that comprises a user interface (UI) 2340 forproviding, for example, results of sequencing analysis, etc. Examples ofUIs include, without limitation, a graphical user interface (GUI) andweb-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 2305. Thealgorithm can, for example, perform sequencing, etc.

Devices, systems, compositions and methods of the present disclosure maybe used for various applications, such as, for example, processing asingle analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g.,DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein)form a single cell. For example, a biological particle (e.g., a cell orcell bead) is partitioned in a partition (e.g., droplet), and multipleanalytes from the biological particle are processed for subsequentprocessing. The multiple analytes may be from the single cell. This mayenable, for example, simultaneous proteomic, transcriptomic and genomicanalysis of the cell.

Example 1: Alternate Method of Adding Multiplexing Information to 2-PartProbe Design

Cells can be processed by barcoded probes as described generally inFIGS. 22A-C and the accompanying text. However, instead of including thebarcode directly on the probes, the barcodes can be added posthybridization, but prior to pooling and partition-based barcoding (e.g.,2207). As shown in FIG. 24 , Multiplexing can occur after a non-barcodedligation probe pair is ligated and/or hybridized to the template. Thefree ends of the probe can be barcoded by attachment to barcodemolecules by sticky end ligation, blunt end ligation, single strandedligation, or extension. In some instances, a splint molecule is utilizedto hybridize the barcodes to the probes for, e.g., ligation orextension. The multiplexed cell can now be partitioned and barcoded(e.g., hybridization of the capture sequence to a sequence on a nucleicacid barcode molecule (e.g., 2271)) as described elsewhere. This methodallows the synthesis of a single set of diverse detection probes, andadd the barcoding information using a less expensive reagent (barcodingoligonucleotides compatible with all probes in the pool).

Example 2: Multiplexing Using Padlock Probes

Cells can be processed by barcoded probes as described generally inFIGS. 22A-C and the accompanying text. However, instead of utilizing atwo-probe approach (e.g., 2201 and 2202), a padlock probe (such as thosedescribed in FIG. 13A-B) can be utilized. As shown in FIG. 25 , apadlock probe is annealed to template (RNA or DNA) and properly annealedprobes (can be a DNA or RNA ligase depending on probe design) areligated. Optionally, a rolling circle amplification can be utilized toboost the signal (in which an optional UMI can be included in thepadlock). The probe is then cut and the cut site (cut site could be aspecific sequence, a cleavable moiety like an abasic site or uracil, achemical linker that is cleavable, etc.). The cut molecule is thenpartitioned and barcoded (e.g., hybridization of the capture sequence toa sequence on a nucleic acid barcode molecule (e.g., 2271)) as describedelsewhere. Alternately the cutting can be done concurrently withbarcoding.

In another alternative example, the padlock probe does not comprise abarcode and is annealed to the template molecule and cut at the cutsite. The free ends of the probe can be barcoded by attachment nucleicacid barcode molecules using sticky end ligation, blunt end ligation,single stranded ligation, or extension (as described in Example 1 andFIG. 24 ). In some instances, a splint molecule is utilized to hybridizethe barcodes to the probes for, e.g., ligation or extension. The cellcan now be partitioned and barcoded (e.g., hybridization of the capturesequence to a sequence on a nucleic acid barcode molecule (e.g., 2271))as described elsewhere.

Example 3: Multiplexing in Context of Gap-Fill Reactions (Using Padlocksor Two-Probe Designs)

Cells can be processed by barcoded probes as described generally inFIGS. 22A-C and the accompanying text. However, a gap-fill reaction canbe utilized prior to probe ligation and processing. As shown in FIG. 26, the probes (e.g., padlock or two-probe approach described elsewhereherein) are annealed to a template (RNA or DNA) nucleic acid. Apolymerase (DNA polymerase, reverse transcriptase or RNA polymerase) isuntitled to fill in the space between the probes. Optionally, the 3′ or5′ ends of the probe can be protected from exonuclease activity by aphosphorothioate modification. The gap-fill functionality allows thecapture of sequence-specific information like splicing, fusions, orsequence variants. The multiplexed cell can now be partitioned andbarcoded (e.g., hybridization of the capture sequence to a sequence on anucleic acid barcode molecule (e.g., 2271)) as described elsewhere.

Example 4: Direct Ligation of Fragmented RNA or DNA to Barcoded Handles

Cells can be processed by barcoded probes as described generally inFIGS. 22A-C and the accompanying text. However, as shown in FIG. 27 ,RNA can be detected by directly ligating barcoded (BC1 and BC2) PCRhandles/capture sequences to RNA fragments. This may be done in freshcells, fixed cells, or cell beads. It may include a step of fragmentingthe nucleic acids of interest. After flanking sequences are attached,downstream partitioning and barcoding in partitions is done, followed byamplification (PCR or RT-PCR) and sequencing to identify the capturedsequence and the cell of origin.

EMBODIMENTS

In some cases, the present disclosure provides a method according to thefollowing embodiments:

-   -   1. A method of analyzing a sample comprising a nucleic acid        molecule, comprising:        -   (a) providing:            -   a sample comprising said nucleic acid molecule, wherein                said nucleic acid molecule comprises a first target                region and a second target region, wherein said first                target region and said second target region are disposed                on a same strand of said nucleic acid molecule;            -   (ii) a first probe comprising a first probe sequence and                a second probe sequence, wherein said first probe                sequence of said first probe is complementary to said                first target region of said nucleic acid molecule; and            -   (iii) a second probe comprising a third probe sequence,                wherein said third probe sequence of said second probe                is complementary to said second target region of said                nucleic acid molecule;        -   (b) subjecting said sample to conditions sufficient to (i)            hybridize said first probe sequence of said first probe to            said first target region of said nucleic acid molecule,            and (ii) hybridize said third probe sequence of said second            probe to said second target region of said nucleic acid            molecule to yield a probe-associated nucleic acid molecule;        -   (c) subjecting said probe-associated nucleic acid molecule            to conditions sufficient to yield a probe-linked nucleic            acid molecule comprising said first probe linked to said            second probe; and        -   (d) within a partition, attaching a barcode sequence to said            first probe.    -   2. The method of embodiment 1, wherein said partition is a well        among a plurality of wells.    -   3. The method of embodiment 1, wherein said partition is a        droplet among a plurality of droplets.    -   4. The method of any one of embodiments 1-3, wherein (d)        comprises (i) providing, in said partition, a nucleic acid        barcode molecule comprising a binding sequence and a barcode        sequence, wherein said binding sequence is complementary to said        second probe sequence of said first probe, and (ii) subjecting        said partition to conditions sufficient to hybridize said        binding sequence to said second probe sequence.    -   5. The method of embodiment 4, further comprising subjecting        said partition to conditions sufficient to extend said second        probe sequence hybridized to said binding sequence of said        nucleic acid barcode molecule to generate an extended first        probe, wherein the extended first probe comprises a sequence        complementary to said barcode sequence.    -   6. The method of embodiment 5, further comprising subjecting        said extended first probe hybridized to said nucleic acid        barcode molecule to conditions sufficient to separate said        nucleic acid barcode molecule from said extended first probe.    -   7. The method of embodiment 5 or 6, further comprising        subjecting said extended first probe hybridized to said nucleic        acid barcode molecule to conditions sufficient to conduct an        amplification reaction to generate an amplification product,        which amplification product comprises said barcode sequence or a        complement thereof    -   8. The method of embodiment 7, wherein said amplification        reaction is a polymerase chain reaction.    -   9. The method of embodiment 7 or 8, wherein said amplification        reaction is performed within said partition.    -   10. The method of embodiment 9, further comprising recovering        said amplification product from said partition.    -   11. The method of embodiment 7 or 8, wherein said amplification        reaction is performed outside of said partition.    -   12. The method of embodiment 10 or 11, further comprising        sequencing said amplification product.    -   13. The method of any one of embodiments 1-3, further        comprising (i) providing a splint oligonucleotide comprising a        first sequence that is complementary to said second probe        sequence and a second sequence, and (ii) subjecting said        partition to conditions sufficient to hybridize said first        sequence of said splint oligonucleotide to said second probe        sequence of said first probe.    -   14. The method of embodiment 13, wherein said first sequence of        said splint oligonucleotide hybridizes to said second probe        sequence of said first probe prior to (c).    -   15. The method of embodiment 13, wherein said first sequence of        said splint oligonucleotide hybridizes to said second probe        sequence of said first probe after (c).    -   16. The method of embodiment 13, wherein said first sequence of        said splint oligonucleotide hybridizes to said second probe        sequence of said first probe prior to (d).    -   17. The method of any one of embodiments 13-16, wherein (d)        comprises (i) providing, in said partition, a nucleic acid        barcode molecule comprising a binding sequence and a barcode        sequence, wherein said binding sequence is complementary to said        second sequence of said splint oligonucleotide, and (ii)        subjecting said partition to conditions sufficient to hybridize        said binding sequence to said second sequence of said splint        oligonucleotide.    -   18. The method of embodiment 17, wherein said binding sequence        of said nucleic acid barcode molecule comprises ribobases.    -   19. The method of embodiment 17 or 18, further comprising        subjecting said splint oligonucleotide hybridized to said first        probe and said nucleic acid barcode molecule to conditions        sufficient to ligate said second probe sequence hybridized to        said splint oligonucleotide to said binding sequence of said        nucleic acid barcode molecule.    -   20. The method of any one of embodiments 17-19, further        comprising subjecting said splint oligonucleotide hybridized to        said first probe and said nucleic acid barcode molecule to        conditions sufficient to extend said second sequence of said        splint oligonucleotide to an end of said nucleic acid barcode        molecule to generate an extended splint oligonucleotide, wherein        the extended splint oligonucleotide comprises a sequence        complementary to said barcode sequence.    -   21. The method of embodiment 20, further comprising subjecting        said extended splint oligonucleotide hybridized to said first        probe and said nucleic acid barcode molecule to conditions        sufficient to separate said extended splint oligonucleotide from        said first probe and said nucleic acid barcode molecule.    -   22. The method of any one of embodiments 17-19, further        comprising subjecting said splint oligonucleotide hybridized to        said first probe and said nucleic acid barcode molecule to        conditions sufficient to extend said first sequence of said        splint oligonucleotide to generate an extended nucleic acid        barcode product, wherein said extended nucleic acid barcode        product comprises a sequence complementary to said first probe        sequence of said first probe and a sequence complementary to        said third probe sequence of said second probe.    -   23. The method of any one of embodiments 19-21, further        comprising subjecting said first probe and said nucleic acid        barcode molecule to conditions sufficient to conduct an        amplification reaction to generate an amplification product,        which amplification product comprises said barcode sequence or a        complement thereof and said first probe sequence or a complement        thereof    -   24. The method of embodiment 23, wherein said amplification        reaction is a polymerase chain reaction.    -   25. The method of embodiment 23 or 24, wherein said        amplification reaction is performed within said partition.    -   26. The method of embodiment 25, further comprising recovering        said amplification product from said partition.    -   27. The method of embodiment 23 or 24, wherein said        amplification reaction is performed outside of said partition.    -   28. The method of embodiment 26 or 27, further comprising        sequencing said amplification product.    -   29. The method of any one of embodiments 4-28, wherein said        nucleic acid barcode molecule further comprises a unique        molecular identifier sequence.    -   30. The method of any one of embodiments 4-29, wherein said        nucleic acid barcode molecule further comprises a sequencing        primer.    -   31. The method of any one of embodiments 4-30, wherein,        subsequent to (c), said probe-associated nucleic acid molecule        is co-partitioned with said nucleic acid barcode molecule.    -   32. The method of any one of embodiments 4-30, wherein,        subsequent to (a), said nucleic acid molecule is co-partitioned        with said first probe, said second probe, and said nucleic acid        barcode molecule.    -   33. The method of embodiment 32, wherein (c) is performed within        said partition.    -   34. The method of embodiment 33, wherein (b) and (c) are        performed within said partition.    -   35. The method of any one of embodiments 4-34, wherein said        second probe comprises a fourth probe sequence, and wherein said        method further comprises providing a nucleic acid binding        molecule in said partition, wherein said nucleic acid binding        molecule comprises a second binding sequence that is        complementary to said fourth probe sequence of said second        probe.    -   36. The method of embodiment 35, wherein said nucleic acid        binding molecule further comprises a third binding sequence.    -   37. The method of embodiment 35 or 36, wherein said nucleic acid        binding molecule further comprises a second barcode sequence.    -   38. The method of any one of embodiments 35-37, further        comprising hybridizing said second binding sequence to said        fourth probe sequence of said second probe within said        partition.    -   39. The method of any one of embodiments 4-38, wherein said        nucleic acid barcode molecule is coupled to a bead.    -   40. The method of embodiment 39, wherein said bead is a gel        bead.    -   41. The method of embodiment 39 or 40, wherein said nucleic acid        barcode molecule is coupled to said bead via a labile moiety.    -   42. The method of any one of embodiments 39-41, wherein said        bead comprises a plurality of nucleic acid barcode molecules        coupled thereto, wherein said plurality of nucleic acid barcode        molecules comprise said nucleic acid barcode molecule.    -   43. The method of embodiment 42, wherein said bead comprises at        least 10,000 nucleic acid barcode molecules coupled thereto.    -   44. The method of embodiment 43, wherein said bead comprises at        least 100,000 nucleic acid barcode molecules coupled thereto.    -   45. The method of embodiment 44, wherein said bead comprises at        least 1,000,000 nucleic acid barcode molecules coupled thereto.    -   46. The method of embodiment 45, wherein said bead comprises at        least 10,000,000 nucleic acid barcode molecules coupled thereto.    -   47. The method of any one of embodiments 42-46, wherein said        plurality of nucleic acid barcode molecules are releasably        coupled to said bead.    -   48. The method of embodiment 47, wherein said plurality of        nucleic acid barcode molecules are releasable from said bead        upon application of a stimulus.    -   49. The method of embodiment 48, wherein said stimulus is        selected from the group consisting of a thermal stimulus, a        photo stimulus, and a chemical stimulus.    -   50. The method of embodiment 49, wherein said stimulus is a        reducing agent.    -   51. The method of embodiment 50, wherein said stimulus is        dithiothreitol.    -   52. The method of any one of embodiments 48-51, wherein the        application of said stimulus results in one or more of (i)        cleavage of a linkage between nucleic acid barcode molecules of        said plurality of nucleic acid barcode molecules and said bead,        and (ii) degradation of said bead to release nucleic acid        barcode molecules of said plurality of nucleic acid barcode        molecules from said bead.    -   53. The method of any one of embodiments 47-52, wherein said        bead is provided in said partition, and wherein said nucleic        acid barcode molecule is released from said bead within said        partition.    -   54. The method of any one of embodiments 1-53, wherein (c) is        performed before (d).    -   55. The method of any one of embodiments 1-53, wherein (d) is        performed before (c).    -   56. The method of any one of embodiments 1-55, wherein said        first probe further comprises a barcode sequence or unique        molecular identifier.    -   57. The method of any one of embodiments 1-56, wherein said        second probe further comprises a barcode sequence or a unique        molecular identifier.    -   58. The method of any one of embodiments 1-57, wherein said        second probe comprises a fourth probe sequence, which fourth        probe sequence hybridizes to a third target region of said        nucleic acid molecule.    -   59. The method of embodiment 58, wherein said second target        region is not adjacent to said third target region, and wherein        said third probe sequence and said fourth probe sequence of said        second probe are separated by a linker sequence.    -   60. The method of any one of embodiments 1-59, wherein said        first probe sequence of said first probe comprises a first        reactive moiety and said third probe sequence of said second        probe comprises a second reactive moiety, wherein, subsequent to        (b), said first reactive moiety is adjacent to said second        reactive moiety.    -   61. The method of embodiment 60, wherein (c) comprises        subjecting said first reactive moiety and said second reactive        moiety to conditions sufficient to link said first probe        sequence to said third probe sequence.    -   62. The method of embodiment 60 or 61, wherein said first        reactive moiety of said first probe comprises an azide moiety.    -   63. The method of any one of embodiments 60-62, wherein said        second reactive moiety of said second probe comprises an alkyne        moiety.    -   64. The method of any one of embodiments 60-63, wherein said        first probe is linked to said second probe in said probe-linked        nucleic acid molecule via a linker, wherein said linker        comprises a triazole moiety.    -   65. The method of embodiment 60 or 61, wherein said first        reactive moiety of said first probe comprises a phosphorothioate        moiety.    -   66. The method of any one of embodiments 60, 61, or 65, wherein        said second reactive moiety of said second probe comprises an        iodide moiety.    -   67. The method of any one of embodiments 60, 61, 65, or 66,        wherein said first probe is linked to said second probe in said        probe-linked nucleic acid molecule via a linker, wherein said        linker comprises a phosphorothioate bond.    -   68. The method of embodiment 60 or 61, wherein said first        reactive moiety of said first probe comprises an amine moiety.    -   69. The method of any one of embodiments 60, 61, or 68, wherein        said second reactive moiety of said second probe comprises a        phosphate moiety.    -   70. The method of any one of embodiments 60, 61, 68, or 69,        wherein said first probe is linked to said second probe in said        probe-linked nucleic acid molecule via a linker, wherein said        linker comprises a phosphoroamidatephosphoramidate bond.    -   71. The method of any one of embodiments 1-59, wherein (c)        comprises performing a nucleic acid reaction.    -   72. The method of any one of embodiments 1-59 or 71, wherein (c)        comprises performing an enzymatic ligation reaction or an        extension reaction.    -   73. The method of embodiment 72, wherein (c) comprises        performing both an enzymatic ligation reaction and an extension        reaction.    -   74. The method of embodiment 72 or 73, wherein said enzymatic        ligation reaction and/or said extension reaction comprises use        of an enzyme selected from the group consisting of T4 RNL2,        SplintR, T4 DNA ligase, KOD ligase, PBCV1, DNA polymerase, and        Mu polymerase, or a derivative thereof    -   75. The method of any one of embodiments 1-74, wherein, prior to        (a), said first probe is linked to said second probe via one or        more linking sequences.    -   76. The method of embodiment 75, wherein said one or more        linking sequences comprise a spacer sequence.    -   77. The method of embodiment 75 or 76, wherein said one or more        linking sequences comprise a sequencing primer or complement        thereof    -   78. The method of any one of embodiments 75-77, wherein said one        or more linking sequences comprise a unique molecular identifier        sequence.    -   79. The method of any one of embodiments 75-78, wherein said one        or more linking sequences comprise a restriction site.    -   80. The method of any one of embodiments 75-79, wherein said one        or more linking sequences comprise a capture sequence.    -   81. The method of any one of embodiments 75-80, wherein said one        or more linking sequences comprise a thermolabile,        photocleavable, or enzymatically cleavable site.    -   82. The method of any one of embodiments 75-81, wherein said one        or more linking sequences comprise a transposition site.    -   83. The method of any one of embodiments 1-82, wherein said        first target region is adjacent to said second target region.    -   84. The method of any one of embodiments 1-82, wherein said        first target region and said second target region are separated        by a gap region disposed between said first target region and        said second target region.    -   85. The method of embodiment 84, wherein said gap region is at        least one nucleotide long.    -   86. The method of embodiment 84 or 85, wherein said gap region        is between 1-10 nucleotides long.    -   87. The method of embodiment 84 or 85, wherein said gap region        is at least 10 nucleotides long.    -   88. The method of embodiment 87, wherein said gap region is at        least 50 nucleotides long.    -   89. The method of embodiment 88, wherein said gap region is at        least 100 nucleotides long.    -   90. The method of embodiment 89, wherein said gap region is at        least 200 nucleotides long.    -   91. The method of embodiment 90, wherein said gap region is at        least 500 nucleotides long.    -   92. The method of embodiment 87, wherein said gap region is        between 50 and 200 nucleotides long.    -   93. The method of any one of embodiments 1-92, further        comprising digesting one or more nucleic acid molecules or        portions thereof using an exonuclease.    -   94. The method of any one of embodiments 1-93, wherein said        first probe or said second probe comprises a known sequence.    -   95. The method of any one of embodiments 1-94, wherein said        first probe or said second probe comprises a degenerate        sequence.    -   96. The method of any one of embodiments 1-95, wherein said        first probe or said second probe comprises a Phi-29 based        rolling circle amplification sequence.    -   97. The method of any one of embodiments 1-96, wherein said        first probe or said second probe comprises a cleavable site,        wherein said cleavable site is cleavable using a thermal,        photo-, chemical, or biological stimulus.    -   98. The method of any one of embodiments 1-97, wherein said        first probe or said second probe comprises a transposition site.    -   99. The method of any one of embodiments 1-98, wherein said        sample comprises a cell, and wherein said nucleic acid molecule        is contained within said cell.    -   100. The method of embodiment 99, further comprising, subsequent        to (a), permeabilizing said cell, thereby providing access to        said nucleic acid molecule.    -   101. The method of embodiment 99, further comprising, subsequent        to (a), lysing said cell, thereby releasing said nucleic acid        molecule from said cell.    -   102. The method of any one of embodiments 99-101, wherein said        cell is a prokaryotic cell.    -   103. The method of any one of embodiments 99-101, wherein said        cell is a eukaryotic cell.    -   104. The method of any one of embodiments 99-101, wherein said        cell is a lymphocyte.    -   105. The method of any one of embodiments 99-101, wherein said        cell is a B cell.    -   106. The method of any one of embodiments 99-101, wherein said        cell is a T cell.    -   107. The method of any one of embodiments 99-101, wherein said        cell is a human cell.    -   108. The method of any one of embodiments 99-107, wherein said        cell is a fixed suspension cell or a formalin-fixed        paraffin-embedded cell.    -   109. The method of any one of embodiments 99-108, wherein said        cell is provided within said partition.    -   110. The method of any one of embodiments 1-109, wherein said        nucleic acid molecule is a single-stranded nucleic acid        molecule.    -   111. The method of any one of embodiments 1-110, wherein said        nucleic acid molecule is a ribonucleic acid (RNA) molecule.    -   112. The method of embodiment 111, wherein said nucleic acid        molecule is a messenger RNA (mRNA) molecule.    -   113. The method of embodiment 111 or 112, wherein said nucleic        acid molecule comprises a polyA sequence at a terminus of said        nucleic acid molecule.    -   114. The method of embodiment 111, wherein said RNA molecule        does not comprise a polyA sequence.    -   115. The method of any one of embodiments 111-114, wherein said        nucleic acid molecule comprises an untranslated region (UTR).    -   116. The method of any one of embodiments 111-115, wherein said        nucleic acid molecule comprises a 5′ cap structure.    -   117. The method of any one of embodiments 1-110, wherein said        nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule.    -   118. The method of any one of embodiments 1-117, wherein said        partition further comprises one or more reagents selected from        the group consisting of fluorophores, oligonucleotides, primers,        nucleic acid barcode molecules, barcodes, buffers,        deoxynucleotide triphosphates, DNA splints, detergents, reducing        agents, chelating agents, oxidizing agents, nanoparticles,        antibodies, and enzymes.    -   119. The method of any one of embodiments 1-118, wherein said        partition further comprises one or more reagents selected from        the group consisting of temperature-sensitive enzymes,        pH-sensitive enzymes, light-sensitive enzymes, proteases,        ligase, polymerases, reverse transcriptases, restriction        enzymes, nucleases, protease inhibitors, and nuclease        inhibitors.    -   120. The method of embodiment 119, wherein said polymerase is a        polymerase selected from the group of DNA polymerase, RNA        polymerase, Hot Start polymerase, and Warm start polymerase.    -   121. The method of any one of embodiments 1-120, wherein said        sample comprises a cell bead, and wherein said nucleic acid        molecule is contained within said cell bead.    -   122. The method of any one of embodiments 1-121, wherein (a)-(c)        are performed without reverse transcription.    -   123. A method of analyzing a sample comprising a nucleic acid        molecule, comprising:        -   (a) providing:            -   (i) a sample comprising said nucleic acid molecule,                wherein said nucleic acid molecule comprises a target                region;            -   (ii) a probe comprising a probe sequence and a binding                sequence, wherein said probe sequence is complementary                to said target region; and            -   (iii) an adapter comprising a first sequence and a                second sequence, wherein said first sequence of said                adapter is complementary to said binding sequence of                said probe;        -   (b) subjecting said sample to conditions sufficient to            hybridize (i) said probe sequence of said probe to said            target region, and (ii) said binding sequence of said probe            to said first sequence of said adapter, to yield an            adapter-bound probe; and        -   (c) within a partition, barcoding said adapter-bound probe            to provide a barcoded nucleic acid molecule.    -   124. The method of embodiment 123, wherein said partition is a        well among a plurality of wells.    -   125. The method of embodiment 123, wherein said partition is a        droplet among a plurality of droplets.    -   126. The method of any one of embodiments 123-125, wherein said        first sequence of said adapter hybridizes to said binding        sequence of said probe within said partition.    -   127. The method of any one of embodiments 123-125, wherein said        first sequence of said adapter hybridizes to said binding        sequence of said probe outside of said partition.    -   128. The method of any one of embodiments 123-127, wherein (c)        comprises (i) providing, in said partition, a nucleic acid        barcode molecule comprising an overhang sequence and a barcode        sequence, wherein said overhang sequence is complementary to        said second sequence of said adapter, and (ii) subjecting said        partition to conditions sufficient to hybridize said overhang        sequence to said second sequence of said adapter to yield said        barcoded nucleic acid molecule.    -   129. The method of embodiment 128, wherein said overhang        sequence of said nucleic acid barcode molecule comprises        ribobases.    -   130. The method of embodiment 128 or 129, further comprising        subjecting said barcoded nucleic acid molecule to conditions        sufficient to ligate said binding sequence hybridized to said        adapter to said overhang sequence of said nucleic acid barcode        molecule.    -   131. The method of embodiment 130, wherein said ligating occurs        outside of said partition.    -   132. The method of any one of embodiments 128-131, further        comprising subjecting said barcoded nucleic acid molecule to        conditions sufficient to extend said second sequence of said        adapter to an end of said nucleic acid barcode molecule to        generate an extended adapter, wherein the extended adapter        comprises a sequence complementary to said barcode sequence.    -   133. The method of embodiment 132, further comprising subjecting        said extended adapter hybridized to said probe and said nucleic        acid barcode molecule to conditions sufficient to separate said        extended adapter from said probe and said nucleic acid barcode        molecule.    -   134. The method of any one of embodiments 128-134, wherein said        nucleic acid barcode molecule further comprises a unique        molecular identifier sequence.    -   135. The method of any one of embodiments 128-135, wherein said        nucleic acid barcode molecule further comprises a sequencing        primer.    -   136. The method of any one of embodiments 128-135, wherein said        nucleic acid barcode molecule is coupled to a bead.    -   137. The method of embodiment 136, wherein said bead is a gel        bead.    -   138. The method of embodiment 136 or 137, wherein said nucleic        acid barcode molecule is coupled to said bead via a labile        moiety.    -   139. The method of any one of embodiments 136-138, wherein said        bead comprises a plurality of nucleic acid barcode molecules        coupled thereto, wherein said plurality of nucleic acid barcode        molecules comprise said nucleic acid barcode molecule.    -   140. The method of embodiment 139, wherein said bead comprises        at least 10,000 nucleic acid barcode molecules coupled thereto.    -   141. The method of embodiment 140, wherein said bead comprises        at least 100,000 nucleic acid barcode molecules coupled thereto.    -   142. The method of embodiment 141, wherein said bead comprises        at least 1,000,000 nucleic acid barcode molecules coupled        thereto.    -   143. The method of embodiment 142, wherein said bead comprises        at least 10,000,000 nucleic acid barcode molecules coupled        thereto.    -   144. The method of any one of embodiments 136-143, wherein said        plurality of nucleic acid barcode molecules are releasably        coupled to said bead.    -   145. The method of embodiment 144, wherein said plurality of        nucleic acid barcode molecules are releasable from said bead        upon application of a stimulus.    -   146. The method of embodiment 145, wherein said stimulus is        selected from the group consisting of a thermal stimulus, a        photo stimulus, and a chemical stimulus.    -   147. The method of embodiment 146, wherein said stimulus is a        reducing agent.    -   148. The method of embodiment 147, wherein said stimulus is        dithiothreitol.    -   149. The method of any one of embodiments 145-148, wherein the        application of said stimulus results in one or more of (i)        cleavage of a linkage between nucleic acid barcode molecules of        said plurality of nucleic acid barcode molecules and said bead,        and (ii) degradation of said bead to release nucleic acid        barcode molecules of said plurality of nucleic acid barcode        molecules from said bead.    -   150. The method of any one of embodiments 144-149, wherein said        bead is provided in said partition, and wherein said nucleic        acid barcode molecule is released from said bead within said        partition.    -   151. The method of any one of embodiments 123-150, further        comprising recovering said barcoded nucleic acid molecule from        said partition.    -   152. The method of embodiment 151, wherein said partition is a        droplet, and wherein recovering said barcoded nucleic acid        molecule from said partition comprises breaking or bursting said        droplet.    -   153. The method of any one of embodiments 123-153, further        comprising digesting one or more nucleic acid molecules using an        exonuclease.    -   154. The method of embodiment 153, wherein said digesting is        performed after (c) in a bulk solution.    -   155. The method of embodiment 154, wherein said one or more        nucleic acid molecules are probe and adapter molecules that are        not coupled to said nucleic acid molecule.    -   156. The method of any one of embodiments 123-155, further        comprising providing an additional probe comprising an        additional probe sequence, which additional probe sequence is        complementary to an additional target region of said nucleic        acid molecule.    -   157. The method of embodiment 156, wherein said additional        target region is adjacent to said target region of said nucleic        acid molecule.    -   158. The method of embodiment 156, wherein said target region        and said additional target region are disposed on a same strand        of said nucleic acid molecule but are separated by a gap region.    -   159. The method of embodiment 158, wherein said gap region is at        least one nucleotide long.    -   160. The method of embodiment 158 or 159, wherein said gap        region is between 1-10 nucleotides long.    -   161. The method of embodiment 158 or 159, wherein said gap        region is at least 10 nucleotides long.    -   162. The method of embodiment 161, wherein said gap region is at        least 50 nucleotides long.    -   163. The method of embodiment 162, wherein said gap region is at        least 100 nucleotides long.    -   164. The method of embodiment 163, wherein said gap region is at        least 200 nucleotides long.    -   165. The method of embodiment 164, wherein said gap region is at        least 500 nucleotides long.    -   166. The method of embodiment 162, wherein said gap region is        between 50 and 200 nucleotides long.    -   167. The method of any one of embodiments 156-166, wherein said        additional probe further comprises a sequencing primer.    -   168. The method of any one of embodiments 156-167, further        comprising (d) subjecting said barcoded nucleic acid molecule        hybridized to said target region of said nucleic acid molecule        to conditions sufficient to hybridize said additional probe        sequence of said additional probe to said additional target        region.    -   169. The method of embodiment 168, wherein said probe sequence        of said probe comprises a first reactive moiety and said        additional probe sequence of said additional probe comprises a        second reactive moiety, wherein, subsequent to (d), said first        reactive moiety is adjacent to said second reactive moiety.    -   170. The method of embodiment 169, further comprising subjecting        said first reactive moiety and said second reactive moiety to        conditions sufficient to link said probe sequence to said        additional probe sequence.    -   171. The method of embodiment 169 or 170, wherein said first        reactive moiety of said first probe comprises an azide moiety.    -   172. The method of any one of embodiments 169-171, wherein said        second reactive moiety of said second probe comprises an alkyne        moiety.    -   173. The method of any one of embodiments 169-172, wherein said        first probe is linked to said second probe in said probe-linked        nucleic acid molecule via a linker, wherein said linker        comprises a triazole moiety.    -   174. The method of embodiment 169 or 170, wherein said first        reactive moiety of said first probe comprises a phosphorothioate        moiety.    -   175. The method of any one of embodiments 169, 170, or 174,        wherein said second reactive moiety of said second probe        comprises an iodide moiety.    -   176. The method of any one of embodiments 169, 170, 174, or 175,        wherein said first probe is linked to said second probe in said        probe-linked nucleic acid molecule via a linker, wherein said        linker comprises a phosphorothioate bond.    -   177. The method of embodiment 169 or 170, wherein said first        reactive moiety of said first probe comprises an amine moiety.    -   178. The method of any one of embodiments 169, 170, or 177,        wherein said second reactive moiety of said second probe        comprises a phosphate moiety.    -   179. The method of any one of embodiments 169, 170, 177, or 178,        wherein said first probe is linked to said second probe in said        probe-linked nucleic acid molecule via a linker, wherein said        linker comprises a phosphoroamidatephosphoramidate bond.    -   180. The method of embodiment 168, wherein said probe hybridized        to said target region is linked to said additional probe        hybridized to said additional target region via a nucleic acid        reaction.    -   181. The method of embodiment 168, wherein said probe hybridized        to said target region is linked to said additional probe        hybridized to said additional target region via an enzymatic        ligation reaction or an extension reaction.    -   182. The method of embodiment 181, wherein said probe hybridized        to said target region is linked to said additional probe        hybridized to said additional target region via both an        enzymatic ligation reaction and an extension reaction.    -   183. The method of embodiment 181 or 182, wherein said enzymatic        ligation reaction and/or said extension reaction comprises use        of an enzyme selected from the group consisting of T4 RNL2,        SplintR, T4 DNA ligase, KOD ligase, PBCV1, DNA polymerase, and        Mu polymerase, or a derivative thereof    -   184. The method of any one of embodiments 156-183, wherein said        additional probe is provided within said partition.    -   185. The method of any one of embodiments 156-183, wherein said        additional probe is provided within another partition.    -   186. The method of any one of embodiments 168-185, further        comprising subjecting said barcoded nucleic acid molecule        hybridized to said nucleic acid molecule to conditions        sufficient to conduct an amplification reaction to generate an        amplification product, which amplification product comprises        said barcode sequence or a complement thereof and said probe        sequence or a complement thereof    -   187. The method of embodiment 186, wherein said amplification        reaction is a polymerase chain reaction.    -   188. The method of embodiment 186 or 187, wherein said        amplification reaction is performed within said partition.    -   189. The method of embodiment 188, further comprising recovering        said amplification product from said partition.    -   190. The method of embodiment 186 or 187, wherein said        amplification reaction is performed outside of said partition.    -   191. The method of embodiment 189 or 190, further comprising        sequencing said amplification product.    -   192. The method of any one of embodiments 123-191, wherein said        probe further comprises a barcode sequence or unique molecular        identifier.    -   193. The method of any one of embodiments 156-192, wherein said        probe or said additional probe comprises a known sequence.    -   194. The method of any one of embodiments 156-193, wherein said        probe or said additional probe comprises a degenerate sequence.    -   195. The method of any one of embodiments 156-194, wherein said        probe or said additional probe comprises a cleavable site,        wherein said cleavable site is cleavable using a thermal,        photo-, chemical, or biological stimulus.    -   196. The method of any one of embodiments 156-195, wherein said        first probe or said second probe comprises a transposition site.    -   197. The method of any one of embodiments 123-196, wherein said        sample comprises a cell, and wherein said nucleic acid molecule        is contained within said cell.    -   198. The method of embodiment 197, further comprising,        subsequent to (a), permeabilizing said cell, thereby providing        access to said nucleic acid molecule.    -   199. The method of embodiment 197, further comprising,        subsequent to (a), lysing said cell, thereby releasing said        nucleic acid molecule from said cell.    -   200. The method of any one of embodiments 197-199, wherein said        cell is a prokaryotic cell.    -   201. The method of any one of embodiments 197-199, wherein said        cell is a eukaryotic cell.    -   202. The method of any one of embodiments 197-199, wherein said        cell is a lymphocyte.    -   203. The method of any one of embodiments 197-199, wherein said        cell is a B cell.    -   204. The method of any one of embodiments 197-199, wherein said        cell is a T cell.    -   205. The method of any one of embodiments 197-199, wherein said        cell is a human cell.    -   206. The method of any one of embodiments 197-205, wherein said        cell is a fixed suspension cell or a formalin-fixed        paraffin-embedded cell.    -   207. The method of any one of embodiments 197-206, wherein said        cell is provided within said partition.    -   208. The method of any one of embodiments 123-207, wherein said        nucleic acid molecule is a single-stranded nucleic acid        molecule.    -   209. The method of any one of embodiments 123-208, wherein said        nucleic acid molecule is a ribonucleic acid (RNA) molecule.    -   210. The method of embodiment 209, wherein said nucleic acid        molecule is a messenger RNA (mRNA) molecule.    -   211. The method of embodiment 209 or 210, wherein said nucleic        acid molecule comprises a polyA sequence at a terminus of said        nucleic acid molecule.    -   212. The method of embodiment 209, wherein said RNA molecule        does not comprise a polyA sequence.    -   213. The method of any one of embodiments 209-212, wherein said        nucleic acid molecule comprises an untranslated region (UTR).    -   214. The method of any one of embodiments 209-213, wherein said        nucleic acid molecule comprises a 5′ cap structure.    -   215. The method of any one of embodiments 123-208, wherein said        nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule.    -   216. The method of any one of embodiments 123-215, wherein said        partition further comprises one or more reagents selected from        the group consisting of fluorophores, oligonucleotides, primers,        nucleic acid barcode molecules, barcodes, buffers,        deoxynucleotide triphosphates, DNA splints, detergents, reducing        agents, chelating agents, oxidizing agents, nanoparticles,        antibodies, and enzymes.    -   217. The method of any one of embodiments 123-216, wherein said        partition further comprises one or more reagents selected from        the group consisting of temperature-sensitive enzymes,        pH-sensitive enzymes, light-sensitive enzymes, proteases,        ligase, polymerases, reverse transcriptases, restriction        enzymes, nucleases, protease inhibitors, and nuclease        inhibitors.    -   218. The method of embodiment 217, wherein said polymerase is a        polymerase selected from the group of DNA polymerase, RNA        polymerase, Hot Start polymerase, and Warm start polymerase    -   219. The method of any one of embodiments 123-218, wherein said        sample comprises a cell bead, and wherein said nucleic acid        molecule is contained within said cell bead.    -   220. The method of any one of embodiments 123-219, wherein        (a)-(c) are performed without reverse transcription.    -   221. A method of analyzing a sample comprising a nucleic acid        molecule, comprising:        -   (a) providing:            -   (i) a sample comprising said nucleic acid molecule,                wherein said nucleic acid molecule comprises a target                region;            -   (ii) a probe comprising a probe sequence and a first                reactive moiety, wherein said probe sequence is                complementary to said target region; and            -   (iii) a nucleic acid barcode molecule comprising a                second reactive moiety and a barcode sequence;        -   (b) subjecting said sample to conditions sufficient to            hybridize said probe sequence of said probe to said target            region to provide a probe-associated nucleic acid molecule;            and        -   (c) within a partition, subjecting said first reactive            moiety of said probe-associated nucleic acid molecule and            said second reactive moiety of said nucleic acid barcode            molecule to conditions sufficient to link said            probe-associated nucleic acid molecule and said nucleic acid            barcode molecule to provide a barcoded nucleic acid product.    -   222. The method of embodiment 221, wherein said partition is a        well among a plurality of wells.    -   223. The method of embodiment 221, wherein said partition is a        droplet among a plurality of droplets.    -   224. The method of any one of embodiments 221-223, wherein said        probe further comprises a sequencing primer.    -   225. The method of any one of embodiments 221-224, wherein said        probe further comprises a spacer sequence disposed between said        probe sequence and said first reactive moiety.    -   226. The method of any one of embodiments 221-225, wherein (b)        is performed within said partition.    -   227. The method of any one of embodiments 221-225, wherein (b)        is performed outside of said partition.    -   228. The method of any one of embodiments 221-227, wherein said        nucleic acid barcode molecule comprises a unique molecular        identifier sequence.    -   229. The method of any one of embodiments 221-228, wherein said        nucleic acid barcode molecule comprises a sequencing primer.    -   230. The method of any one of embodiments 221-229, wherein said        nucleic acid barcode molecule comprises a spacer sequence        disposed between said second reactive moiety and another        sequence of said nucleic acid barcode molecule.    -   231. The method of any one of embodiments 221-230, wherein said        nucleic acid barcode molecule is coupled to a bead.    -   232. The method of embodiment 231, wherein said bead is a gel        bead.    -   233. The method of embodiment 231 or 232, wherein said nucleic        acid barcode molecule is coupled to said bead via a labile        moiety.    -   234. The method of any one of embodiments 231-233, wherein said        bead comprises a plurality of nucleic acid barcode molecules        coupled thereto, wherein said plurality of nucleic acid barcode        molecules comprise said nucleic acid barcode molecule.    -   235. The method of embodiment 234, wherein said bead comprises        at least 10,000 nucleic acid barcode molecules coupled thereto.    -   236. The method of embodiment 235, wherein said bead comprises        at least 100,000 nucleic acid barcode molecules coupled thereto.    -   237. The method of embodiment 236, wherein said bead comprises        at least 1,000,000 nucleic acid barcode molecules coupled        thereto.    -   238. The method of embodiment 237, wherein said bead comprises        at least 10,000,000 nucleic acid barcode molecules coupled        thereto.    -   239. The method of any one of embodiments 234-238, wherein said        plurality of nucleic acid barcode molecules are releasably        coupled to said bead.    -   240. The method of embodiment 239, wherein said plurality of        nucleic acid barcode molecules are releasable from said bead        upon application of a stimulus.    -   241. The method of embodiment 240, wherein said stimulus is        selected from the group consisting of a thermal stimulus, a        photo stimulus, and a chemical stimulus.    -   242. The method of embodiment 241, wherein said stimulus is a        reducing agent.    -   243. The method of embodiment 242, wherein said stimulus is        dithiothreitol.    -   244. The method of any one of embodiments 240-243, wherein the        application of said stimulus results in one or more of (i)        cleavage of a linkage between nucleic acid barcode molecules of        said plurality of nucleic acid barcode molecules and said bead,        and (ii) degradation of said bead to release nucleic acid        barcode molecules of said plurality of nucleic acid barcode        molecules from said bead.    -   245. The method of any one of embodiments 231-245, wherein said        bead is provided in said partition, and wherein said nucleic        acid barcode molecule is released from said bead within said        partition.    -   246. The method of any one of embodiments 221-245, further        comprising recovering said barcoded nucleic acid molecule from        said partition.    -   247. The method of embodiment 246, wherein said partition is a        droplet, and wherein recovering said barcoded nucleic acid        molecule from said partition comprises breaking or bursting said        droplet.    -   248. The method of any one of embodiments 221-247, wherein said        first reactive moiety of said first probe comprises an azide        moiety.    -   249. The method of any one of embodiments 221-248, wherein said        second reactive moiety of said second probe comprises an alkyne        moiety.    -   250. The method of any one of embodiments 221-249, wherein said        first probe is linked to said second probe in said probe-linked        nucleic acid molecule via a linker, wherein said linker        comprises a triazole moiety.    -   251. The method of any one of embodiments 221-247, wherein said        first reactive moiety of said first probe comprises a        phosphorothioate moiety.    -   252. The method of any one of embodiments 221-247 or 251,        wherein said second reactive moiety of said second probe        comprises an iodide moiety.    -   253. The method of any one of embodiments 221-247, 251, or 252,        wherein said first probe is linked to said second probe in said        probe-linked nucleic acid molecule via a linker, wherein said        linker comprises a phosphorothioate bond.    -   254. The method of any one of embodiments 221-247, wherein said        first reactive moiety of said first probe comprises an amine        moiety.    -   255. The method of any one of embodiments 221-247 or 254,        wherein said second reactive moiety of said second probe        comprises a phosphate moiety.    -   256. The method of any one of embodiments 221-247, 254, or 255,        wherein said first probe is linked to said second probe in said        probe-linked nucleic acid molecule via a linker, wherein said        linker comprises a phosphoroamidatephosphoramidate bond.    -   257. The method of any one of embodiments 221-256 further        comprising subjecting said barcoded nucleic acid product        hybridized to said nucleic acid molecule to conditions        sufficient to conduct an amplification reaction to generate an        amplification product, which amplification product comprises        said barcode sequence or a complement thereof and said probe        sequence or a complement thereof    -   258. The method of embodiment 257, wherein said amplification        reaction is a polymerase chain reaction.    -   259. The method of embodiment 257 or 258, wherein said        amplification reaction is performed within said partition.    -   260. The method of embodiment 259, further comprising recovering        said amplification product from said partition.    -   261. The method of embodiment 257 or 258, wherein said        amplification reaction is performed outside of said partition.    -   262. The method of embodiment 260 or 261, further comprising        sequencing said amplification product.    -   263. The method of any one of embodiments 221-262, wherein said        probe further comprises a barcode sequence or unique molecular        identifier.    -   264. The method of any one of embodiments 221-263, wherein said        probe comprises a known sequence.    -   265. The method of any one of embodiments 221-264, wherein said        probe comprises a degenerate sequence.    -   266. The method of any one of embodiments 221-265, wherein said        probe comprises a cleavable site, wherein said cleavable site is        cleavable using a thermal, photo-, chemical, or biological        stimulus.    -   267. The method of any one of embodiments 221-266, wherein said        probe comprises a transposition site.    -   268. The method of any one of embodiments 221-267, wherein said        sample comprises a cell, and wherein said nucleic acid molecule        is contained within said cell.    -   269. The method of embodiment 268, further comprising,        subsequent to (a), permeabilizing said cell, thereby providing        access to said nucleic acid molecule.    -   270. The method of embodiment 268, further comprising,        subsequent to (a), lysing said cell, thereby releasing said        nucleic acid molecule from said cell.    -   271. The method of any one of embodiments 268-270, wherein said        cell is a prokaryotic cell.    -   272. The method of any one of embodiments 268-270, wherein said        cell is a eukaryotic cell.    -   273. The method of any one of embodiments 268-270, wherein said        cell is a lymphocyte.    -   274. The method of any one of embodiments 268-270, wherein said        cell is a B cell.    -   275. The method of any one of embodiments 268-270, wherein said        cell is a T cell.    -   276. The method of any one of embodiments 268-270, wherein said        cell is a human cell.    -   277. The method of any one of embodiments 268-276, wherein said        cell is a fixed suspension cell or a formalin-fixed        paraffin-embedded cell.    -   278. The method of any one of embodiments 268-277, wherein said        cell is provided within said partition.    -   279. The method of any one of embodiments 221-278, wherein said        nucleic acid molecule is a single-stranded nucleic acid        molecule.    -   280. The method of any one of embodiments 221-279, wherein said        nucleic acid molecule is a ribonucleic acid (RNA) molecule.    -   281. The method of embodiment 280, wherein said nucleic acid        molecule is a messenger RNA (mRNA) molecule.    -   282. The method of embodiment 280 or 281, wherein said nucleic        acid molecule comprises a polyA sequence at a terminus of said        nucleic acid molecule.    -   283. The method of embodiment 280 or 281, wherein said RNA        molecule does not comprise a polyA sequence.    -   284. The method of any one of embodiments 280-283, wherein said        nucleic acid molecule comprises an untranslated region (UTR).    -   285. The method of any one of embodiments 280-284, wherein said        nucleic acid molecule comprises a 5′ cap structure.    -   286. The method of any one of embodiments 221-279, wherein said        nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule.    -   287. The method of any one of embodiments 221-286, wherein said        partition further comprises one or more reagents selected from        the group consisting of fluorophores, oligonucleotides, primers,        nucleic acid barcode molecules, barcodes, buffers,        deoxynucleotide triphosphates, DNA splints, detergents, reducing        agents, chelating agents, oxidizing agents, nanoparticles,        antibodies, and enzymes.    -   288. The method of any one of embodiments 221-287, wherein said        partition further comprises one or more reagents selected from        the group consisting of temperature-sensitive enzymes,        pH-sensitive enzymes, light-sensitive enzymes, proteases,        ligase, polymerases, reverse transcriptases, restriction        enzymes, nucleases, protease inhibitors, and nuclease        inhibitors.    -   289. The method of embodiment 288, wherein said polymerase is a        polymerase selected from the group of DNA polymerase, RNA        polymerase, Hot Start polymerase, and Warm start polymerase    -   290. The method of any one of embodiments 221-289, wherein said        sample comprises a cell bead, and wherein said nucleic acid        molecule is contained within said cell bead.    -   291. The method of any one of embodiments 221-290, wherein        (a)-(c) are performed without reverse transcription.    -   292. The method of any one of embodiments 1-33, wherein (a)-(d)        are repeated for a plurality of nucleic acid molecules, a        plurality of first probes, a plurality of second probes, a        plurality of barcode sequences, and a plurality of partitions,        wherein in (d), a plurality of probe-associated nucleic acid        molecule are partitioned among a plurality of partitions, where        each partition of said plurality of partitions comprises a        different barcode sequence of said plurality of barcode        sequences.    -   293. The method of embodiments 292, wherein (c) comprises        generating a plurality of probe-associated nucleic acid        molecules, and wherein (c) is performed in an additional        plurality of partitions, which additional plurality of        partitions are different than said plurality of partitions.    -   294. The method of embodiment 293, wherein said plurality of        partitions is a plurality of droplets and wherein said plurality        of additional partitions is a plurality of wells.    -   295. The method of embodiment 293 or 294, wherein said plurality        of first probes or said plurality of second probes comprises a        plurality of partition barcode sequences.    -   296. The method of embodiment 295, wherein each probe-associated        nucleic acid molecule of said plurality of probe-associated        nucleic acid molecules generated in said additional plurality of        partitions in (c) comprises a partition barcode sequence of said        plurality of partition barcode sequences.    -   297. The method of embodiment 296, wherein each additional        partition of said additional plurality of partitions comprises a        different partition barcode sequence of said plurality of        partition barcode sequences.    -   298. The method of embodiment 297, further comprising, prior to        (d), recovering said plurality of probe-associated nucleic acid        molecules from said additional plurality of partitions.    -   299. The method of embodiment 298, wherein (d) comprises using a        plurality of nucleic acid barcode molecules comprising said        plurality of barcode sequences to attach said plurality of        barcode sequences to first probes of said plurality of        probe-associated nucleic acid molecules.    -   300. The method of embodiment 299, wherein each partition of        said plurality of partitions comprises a different barcode        sequence of said plurality of barcode sequences.

Additional Embodiments

In some cases, the present disclosure provides a method according to thefollowing additional embodiments:

-   -   1. A method of analyzing a sample comprising a nucleic acid        molecule, comprising:        -   a. providing:            -   (i) a sample comprising said nucleic acid molecule,                wherein said nucleic acid molecule comprises a first                target region and a second target region, wherein said                first target region and said second target region are                disposed on a same strand of said nucleic acid molecule;            -   (ii) a first probe comprising a first probe sequence and                a second probe sequence, wherein said first probe                sequence of said first probe is complementary to said                first target region of said nucleic acid molecule; and            -   (iii) a second probe comprising a third probe sequence,                wherein said third probe sequence of said second probe                is complementary to said second target region of said                nucleic acid molecule;        -   b. subjecting said sample to conditions sufficient to (i)            hybridize said first probe sequence of said first probe to            said first target region of said nucleic acid molecule,            and (ii) hybridize said third probe sequence of said second            probe to said second target region of said nucleic acid            molecule to yield a probe-associated nucleic acid molecule;        -   c. subjecting said probe-associated nucleic acid molecule to            conditions sufficient to yield a probe-linked nucleic acid            molecule comprising said first probe linked to said second            probe; and        -   d. within a partition, attaching a barcode sequence to said            probe-linked nucleic acid molecule.    -   2. The method of embodiment 1, wherein said partition is a well        among a plurality of wells.    -   3. The method of embodiment 1, wherein said partition is a        droplet among a plurality of droplets.    -   4. The method of any one of embodiments 1-3, wherein (d)        comprises (i) providing, in said partition, a nucleic acid        barcode molecule comprising a binding sequence and a barcode        sequence, wherein said binding sequence is complementary to said        second probe sequence of said first probe, and (ii) subjecting        said partition to conditions sufficient to hybridize said        binding sequence to said second probe sequence.    -   5. The method of embodiment 4, further comprising subjecting        said partition to conditions sufficient to conduct a nucleic        acid extension reaction to generate a barcoded nucleic acid        molecule comprising a sequence corresponding to said first        probe, a sequence corresponding to said second probe, and a        sequence corresponding to said barcode sequence.    -   6. The method of embodiment 4, further comprising subjecting        said partition to conditions sufficient to ligate said        probe-linked nucleic acid molecule to said nucleic acid barcode        molecule to generate a barcoded nucleic acid molecule comprising        a sequence corresponding to said first probe, a sequence        corresponding to said second probe, and a sequence corresponding        to said barcode sequence.    -   7. The method of embodiment 5 or 6, further comprising        subjecting said barcoded nucleic acid molecule to conditions        sufficient to conduct an amplification reaction to generate an        amplification product, which amplification product comprises        nucleic acid molecules comprising said sequence corresponding to        said first probe, said sequence corresponding to said second        probe, and said sequence corresponding to said barcode sequence.    -   8. The method of embodiment 7, wherein said amplification        reaction comprises use of a primer comprising one or more        functional sequences and wherein said amplification product        comprises nucleic acid molecules further comprising said one or        more functional sequences.    -   9. The method of any one of embodiments 7 or 8, wherein said        amplification is isothermal amplification.    -   10. The method of any one of embodiments 7-9, wherein said        amplification reaction is performed within said partition.    -   11. The method of embodiment 10, further comprising recovering        said amplification product from said partition.    -   12. The method of any one of embodiments 7-9, wherein said        amplification reaction is performed outside of said partition.    -   13. The method of any one of embodiments 7-12, further        comprising sequencing said amplification product or a derivative        thereof    -   14. The method of any one of embodiments 1-3, further        comprising (i) providing a splint oligonucleotide comprising a        first sequence complementary to said second probe sequence and a        second sequence, and (ii) subjecting said partition to        conditions sufficient to hybridize said first sequence of said        splint oligonucleotide to said second probe sequence.    -   15. The method of embodiment 13, wherein said first sequence of        said splint oligonucleotide hybridizes to said second probe        sequence prior to (c).    -   16. The method of embodiment 13, wherein said first sequence of        said splint oligonucleotide hybridizes to said second probe        sequence after (c).    -   17. The method of any one of embodiments 13-16, wherein (d)        comprises (i) providing, in said partition, a nucleic acid        barcode molecule comprising a binding sequence and a barcode        sequence, wherein said binding sequence is complementary to said        second sequence of said splint oligonucleotide, and (ii)        subjecting said partition to conditions sufficient to hybridize        said binding sequence to said second sequence of said splint        oligonucleotide.    -   18. The method of embodiment 17, wherein said binding sequence        of said nucleic acid barcode molecule comprises one or more        ribobases.    -   19. The method of embodiment 17 or 18, further comprising        subjecting (i) said splint oligonucleotide hybridized to said        second probe sequence and (ii) said nucleic acid barcode        molecule to conditions sufficient to ligate said probe-linked        nucleic acid molecule to said nucleic acid barcode molecule.    -   20. The method of any one of embodiments 17-19, further        comprising subjecting (i) said splint oligonucleotide hybridized        to said second probe sequence and (ii) said nucleic acid barcode        molecule to conditions sufficient to conduct a nucleic acid        extension reaction to generate a barcoded nucleic acid molecule        comprising a sequence corresponding to said first probe, a        sequence corresponding to said second probe, and a sequence        corresponding to said barcode sequence.    -   21. The method of any one of embodiments 19 or 20, further        comprising subjecting said barcoded nucleic acid molecule to        conditions sufficient to conduct an amplification reaction to        generate an amplification product, which amplification product        comprises nucleic acid molecules comprising said sequence        corresponding to said first probe, said sequence corresponding        to said second probe, and said sequence corresponding to said        barcode sequence.    -   22. The method of embodiment 21, wherein said amplification        reaction is a polymerase chain reaction.    -   23. The method of embodiment 21 or 22, wherein said        amplification reaction is performed within said partition.    -   24. The method of embodiment 23, further comprising recovering        said amplification product from said partition.    -   25. The method of embodiment 21 or 22, wherein said        amplification reaction is performed outside of said partition.    -   26. The method of any one of embodiments 21-25, wherein said        amplification reaction is isothermal amplification.    -   27. The method of any one of embodiments 21-26, further        comprising sequencing said amplification product or derivative        thereof    -   28. The method of any one of embodiments 4-27, wherein said        nucleic acid barcode molecule further comprises a unique        molecular identifier sequence, a sequencing primer sequence,        and/or a partial sequencing primer sequence.    -   29. The method of any one of embodiments 4-28, wherein,        subsequent to (c), said probe-associated nucleic acid molecule        is co-partitioned with said nucleic acid barcode molecule.    -   30. The method of any one of embodiments 4-28, wherein,        subsequent to (a), said nucleic acid molecule is co-partitioned        with said first probe, said second probe, and said nucleic acid        barcode molecule.    -   31. The method of embodiment 30, wherein (c) is performed within        said partition.    -   32. The method of embodiment 31, wherein (b) and (c) are        performed within said partition.    -   33. The method of any one of embodiments 4-32, wherein said        second probe comprises a fourth probe sequence, and wherein said        method further comprises providing a nucleic acid binding        molecule in said partition, wherein said nucleic acid binding        molecule comprises a second binding sequence that is        complementary to said fourth probe sequence of said second        probe.    -   34. The method of embodiment 33, further comprising hybridizing        said second binding sequence to said fourth probe sequence of        said second probe within said partition.    -   35. The method of any one of embodiments 4-34, wherein said        nucleic acid barcode molecule is coupled to a bead.    -   36. The method of embodiment 35, wherein said bead is a gel        bead.    -   37. The method of embodiment 35 or 36, wherein said nucleic acid        barcode molecule is coupled to said bead via a labile moiety.    -   38. The method of any one of embodiments 35-37, wherein said        bead comprises a plurality of nucleic acid barcode molecules        coupled thereto, wherein said plurality of nucleic acid barcode        molecules comprise said nucleic acid barcode molecule.    -   39. The method of embodiment 38, wherein said bead comprises at        least 100,000 nucleic acid barcode molecules coupled thereto.    -   40. The method of embodiment 38 or 39, wherein said plurality of        nucleic acid barcode molecules are releasably coupled to said        bead.    -   41. The method of embodiment 40, wherein said plurality of        nucleic acid barcode molecules are releasable from said bead        upon application of a stimulus.    -   42. The method of embodiment 41, wherein said stimulus is        selected from the group consisting of a thermal stimulus, a        photo stimulus, a biological stimulus, and a chemical stimulus.    -   43. The method of embodiment 42, wherein said stimulus is a        reducing agent.    -   44. The method of any one of embodiments 41-43, wherein the        application of said stimulus results in one or more of (i)        cleavage of a linkage between nucleic acid barcode molecules of        said plurality of nucleic acid barcode molecules and said bead,        and (ii) degradation of said bead to release nucleic acid        barcode molecules of said plurality of nucleic acid barcode        molecules from said bead.    -   45. The method of any one of embodiments 35-44, wherein said        bead is provided in said partition, and wherein said nucleic        acid barcode molecule is released from said bead within said        partition.    -   46. The method of any one of embodiments 1-45, wherein (c) is        performed before (d).    -   47. The method of any one of embodiments 1-45, wherein (d) is        performed before (c).    -   48. The method of any one of embodiments 1-47, wherein said        first probe or said second probe further comprises a barcode        sequence or unique molecular identifier.    -   49. The method of any one of embodiments 1-48, wherein said        second probe comprises a fourth probe sequence, which fourth        probe sequence hybridizes to a third target region of said        nucleic acid molecule.    -   50. The method of embodiment 49, wherein said second target        region is not adjacent to said third target region, and wherein        said third probe sequence and said fourth probe sequence of said        second probe are separated by a linker sequence.    -   51. The method of any one of embodiments 1-50, wherein said        first probe sequence of said first probe comprises a first        reactive moiety and said third probe sequence of said second        probe comprises a second reactive moiety, wherein, subsequent to        (b), said first reactive moiety is adjacent to said second        reactive moiety.    -   52. The method of embodiment 51, wherein (c) comprises        subjecting said first reactive moiety and said second reactive        moiety to conditions sufficient to link said first probe        sequence to said third probe sequence.    -   53. The method of embodiment 51 or 52, wherein said first        reactive moiety of said first probe or said second reactive        moiety of said second probe comprises an azide moiety, an alkyne        moiety, a phosphorothioate moiety, an iodide moiety, an amine        moiety, or a phosphate moiety.    -   54. The method of any one of embodiments 51-53, wherein said        first probe is linked to said second probe in said probe-linked        nucleic acid molecule via a linker, wherein said linker        comprises a triazole moiety, a phosphorothioate bond, or a        phosphoroamidatephosphoramidate bond.    -   55. The method of any one of embodiments 1-50, wherein (c)        comprises performing an enzymatic ligation reaction and/or an        extension reaction.    -   56. The method of embodiment 55, wherein said enzymatic ligation        reaction and/or said extension reaction comprises use of an        enzyme selected from the group consisting of T4 RNL2, SplintR,        T4 DNA ligase, KOD ligase, PBCV1, DNA polymerase, and Mu        polymerase, or a derivative thereof    -   57. The method of any one of embodiments 1-56, wherein, prior to        (a), said first probe is linked to said second probe via one or        more linking sequences.    -   58. The method of embodiment 57, wherein said one or more        linking sequences comprise one or more of a spacer sequence, a        sequencing primer or complement thereof, a capture sequence, a        restriction site, a transposition site, and a unique molecular        identifier sequence.    -   59. The method of embodiment 57 or 58, wherein said one or more        linking sequences comprise a thermolabile, photocleavable, or        enzymatically cleavable site.    -   60. The method of any one of embodiments 1-59, wherein said        first target region is adjacent to said second target region.    -   61. The method of any one of embodiments 1-59, wherein said        first target region and said second target region are separated        by a gap region disposed between said first target region and        said second target region.    -   62. The method of embodiment 61, wherein said gap region is at        least one nucleotide long.    -   63. The method of embodiment 62, wherein said gap region is at        least 10 nucleotides long.    -   64. The method of embodiment 63, wherein said gap region is at        least 100 nucleotides long.    -   65. The method of any one of embodiments 1-64, further        comprising digesting one or more nucleic acid molecules or        portions thereof using an exonuclease.    -   66. The method of any one of embodiments 1-65, wherein said        first probe or said second probe comprises a known sequence or a        degenerate sequence.    -   67. The method of any one of embodiments 1-66, wherein said        first probe or said second probe comprises a Phi-29 based        rolling circle amplification sequence.    -   68. The method of any one of embodiments 1-67, wherein said        first probe or said second probe comprises a cleavable site,        wherein said cleavable site is cleavable using a thermal,        photo-, chemical, or biological stimulus.    -   69. The method of any one of embodiments 1-68, further        comprising contacting said first probe or said second probe with        a transposase.    -   70. The method of any one of embodiments 1-69, wherein said        sample comprises a cell, and wherein said nucleic acid molecule        is contained within said cell.    -   71. The method of embodiment 70, further comprising, subsequent        to (a), lysing or permeabilizing said cell, thereby providing        access to said nucleic acid molecule.    -   72. The method of embodiment 70 or 71, wherein said cell is a        prokaryotic cell.    -   73. The method of embodiment 70 or 71, wherein said cell is a        eukaryotic cell.    -   74. The method of embodiment 70 or 71, wherein said cell is a        human cell.    -   75. The method of any one of embodiments 70-74, wherein said        cell is a fixed suspension cell or a formalin-fixed        paraffin-embedded cell.    -   76. The method of any one of embodiments 70-75, wherein said        cell is provided within said partition.    -   77. The method of embodiment 76, wherein said cell is a single        cell.    -   78. The method of any one of embodiments 1-77, wherein said        nucleic acid molecule is a ribonucleic acid (RNA) molecule.    -   79. The method of embodiment 78, wherein said nucleic acid        molecule is a messenger RNA (mRNA) molecule.    -   80. The method of embodiment 78 or 79, wherein said nucleic acid        molecule comprises a poly-A sequence at a terminus of said        nucleic acid molecule.    -   81. The method of any one of embodiments 1-77, wherein said        nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule.    -   82. The method of any one of embodiments 1-81, wherein said        partition further comprises one or more reagents selected from        the group consisting of fluorophores, oligonucleotides, primers,        nucleic acid barcode molecules, barcodes, buffers,        deoxynucleotide triphosphates, DNA splints, detergents, reducing        agents, chelating agents, oxidizing agents, nanoparticles,        antibodies, temperature-sensitive enzymes, pH-sensitive enzymes,        light-sensitive enzymes, proteases, ligases, polymerases,        reverse transcriptases, restriction enzymes, nucleases, protease        inhibitors, and nuclease inhibitors.    -   83. The method of embodiment 82, wherein said polymerase is a        polymerase selected from the group of DNA polymerase, RNA        polymerase, Hot Start polymerase, and Warm start polymerase    -   84. The method of any one of embodiments 1-83, wherein said        sample comprises a cell bead, and wherein said nucleic acid        molecule is contained within said cell bead.    -   85. The method of any one of embodiments 1-84, wherein (a)-(c)        are performed without reverse transcription.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method of analyzing a sample comprising a cellor an isolated cell nucleus comprising a nucleic acid molecule,comprising: (a) providing: (i) the sample comprising the cell or theisolated cell nucleus comprising the nucleic acid molecule, wherein thenucleic acid molecule comprises a first target region and a secondtarget region, and wherein the first target region and the second targetregion are both disposed on a strand of the nucleic acid molecule; (ii)a first probe comprising a first probe sequence and a second probesequence, wherein the first probe sequence of the first probe iscomplementary to the first target region of the nucleic acid molecule;(iii) a second probe comprising a third probe sequence, wherein thethird probe sequence is complementary to the second target region of thenucleic acid molecule; (iv) a barcode oligonucleotide comprising: (1) afirst barcode sequence that identifies the sample and (2) a thirdsequence; (v) a splint oligonucleotide comprising: (A) a first splintsequence complementary to the second probe sequence of the first probe,and (B) a second splint sequence complementary to the third sequence;wherein, in the cell or the isolated cell nucleus: (i) the first probesequence of the first probe is hybridized to the first target region ofthe nucleic acid molecule, (ii) the third probe sequence of the secondprobe is hybridized to the second target region of the nucleic acidmolecule, (iii) the first splint sequence of the splint oligonucleotideis hybridized to the second probe sequence of the first probe, and (iv)the second splint sequence of the splint oligonucleotide is hybridizedto the third sequence of the barcode oligonucleotide; (b) using thefirst probe, the second probe, and the barcode oligonucleotide togenerate an additional nucleic acid molecule comprising the first probe,the second probe, and the barcode oligonucleotide; and (c) using theadditional nucleic acid molecule and a polynucleotide that comprises asecond barcode sequence to generate a barcoded nucleic acid moleculecomprising (i) a sequence corresponding to the first target region andthe second target region, (ii) the first barcode sequence or reversecomplement thereof, and (iii) the second barcode sequence or reversecomplement thereof, wherein the second barcode sequence or reversecomplement thereof identifies the cell or isolated cell nucleus.
 2. Themethod of claim 1, wherein (c) is performed in a partition.
 3. Themethod of claim 2, wherein the partition is selected from a well among aplurality of wells and a droplet among a plurality of droplets.
 4. Themethod of claim 2, wherein (c) comprises (i) providing, in thepartition, the polynucleotide comprising the second barcode sequence anda binding sequence, wherein the binding sequence is complementary to thefirst probe sequence of the first probe or the third probe sequence ofthe second probe, and (ii) subjecting the partition to conditionssufficient to hybridize the binding sequence to the first probe sequenceor the third probe sequence.
 5. The method of claim 4, furthercomprising subjecting the partition to conditions sufficient to conducta nucleic acid extension reaction to generate the barcoded nucleic acidmolecule.
 6. The method of claim 1, wherein the polynucleotide iscoupled to a support.
 7. The method of claim 5, further comprisingsubjecting the barcoded nucleic acid molecule to conditions sufficientto conduct an amplification reaction to generate an amplificationproduct, which amplification product comprises nucleic acid moleculescorresponding to the barcoded nucleic acid molecule.
 8. The method ofclaim 7, wherein the amplification reaction comprises use of a primercomprising one or more functional sequences, and wherein the nucleicacid molecules further comprise the one or more functional sequences. 9.The method of claim 7, wherein the amplification reaction is performedwithin the partition.
 10. The method of claim 9, further comprisingrecovering the amplification product from the partition.
 11. The methodof claim 7, wherein the amplification reaction is performed outside ofthe partition.
 12. The method of claim 1, further comprising sequencingthe barcoded nucleic acid molecule or derivative thereof.
 13. The methodof claim 1, wherein the polynucleotide further comprises a uniquemolecular identifier sequence, a sequencing primer sequence, or apartial sequencing primer sequence.
 14. The method of claim 1, wherein,in (a), a gap region comprising one or more nucleotides is presentbetween: (i) the first probe and the second probe hybridized to thenucleic acid molecule, or (ii) the first probe and the barcodeoligonucleotide.
 15. The method of claim 14, wherein the gap regioncomprises at least 100 nucleotides.
 16. The method of claim 14, whereinthe gap region is filled with nucleotides prior to or during (b). 17.The method of claim 6, wherein the support is a bead.
 18. The method ofclaim 17, wherein the bead is a gel bead.
 19. The method of claim 1,wherein the first barcode sequence and the second barcode sequence aredifferent from one another.
 20. The method of claim 1, wherein the cellor the isolated nucleus is fixed with a fixative.
 21. The method ofclaim 20, wherein the cell or the isolated nucleus is permeabilized. 22.The method of claim 1, wherein the cell or the isolated cell nucleus isderived from a tissue.
 23. The method of claim 22, wherein the tissue isa formalin-fixed paraffin-embedded tissue.
 24. The method of claim 1,further comprising, in (b): (i) linking the first probe to the secondprobe using ligation, or (ii) linking the first probe to the barcodeoligonucleotide using ligation.
 25. The method of claim 4, wherein thesecond barcode sequence identifies the barcoded nucleic acid molecule ashaving been generated in the partition.
 26. The method of claim 17,wherein the polynucleotide is coupled to the bead via a labile moiety.27. The method of claim 1, wherein hybridization of the first probesequence of the first probe to the first target region of the nucleicacid molecule and hybridization of the third probe sequence of thesecond probe to the second target region of the nucleic acid moleculeoccur prior to hybridization of the first splint sequence of the splintoligonucleotide to the second probe sequence of the first probe.
 28. Themethod of claim 1, wherein the barcoded nucleic acid molecule comprisesa third barcode sequence.
 29. The method of claim 1, wherein the secondprobe comprises a third barcode sequence.
 30. The method of claim 1,wherein the first probe or the second probe comprises a loop sequence.